Methods and Compositions for Detecting Receptor Clustering and Activation Events in Single Cells

ABSTRACT

The invention provides methods and compositions for simultaneously detecting the activation state of a plurality of proteins in single cells using flow cytometry. The invention further provides methods and compositions of screening for bioactive agents capable of coordinately modulating the activity of a plurality of proteins in single cells. The methods and compositions can be used to determine the protein activation profile of a cell for predicting or diagnosing a disease state, and for monitoring treatment of a disease state.

This application is a continuation in part of U.S. Ser. No. 10/193,462,filed Jul. 10, 2002, which claims the benefit of the filing date of U.S.Ser. No. 60/304,434, filed Jul. 10, 2001, and U.S. Ser. No. 60/310,141,filed Aug. 2, 2001.

FIELD OF THE INVENTION

The invention relates generally to the field of protein detection usingflow cytometry. More specifically, the invention relates tosimultaneously detecting the clustering and activation states ofreceptor elements in single cells using flow cytometry and, moreparticularly, using polychromatic flow cytometry.

BACKGROUND OF THE INVENTION

Proteins are the major components of cells. The spatiotemporalexpression pattern and the subcellular localization of proteinsdetermines the shape, structure, and function of cells. Proteins areassembled from 20 different amino acids, each with a distinct side chainand chemical property. This provides for enormous variety in thechemical properties of proteins and the activities they exhibit.

Many proteins are dynamically regulated such that their activity isaltered in response to certain intracellular or extracellular cues. Ingeneral, the response to these cues is mediated by a broad class ofproteins termed receptors. While receptors employ a wide variety ofmeans to transduce intracellular and extracellular cues, a generalmechanism involves a receptor binding a ligand, which activates thereceptor to propagate a signal that eventually results in alteredprotein activity within the cell. Because receptors play such anintegral role in transducing intracellular and extracellular signals,they have been the subject of intense biochemical study.

Study of receptor function often requires immobilization of a receptoron a solid support. In Western blot analysis, receptors of interest arefirst separated by electrophoresis and then transferred and immobilizedonto a nitrocellulose or a polyvinylidene difluoride (PVDF) membrane. Inthe phage display screening of a protein expression library, severalhundred thousand proteins expressed by phages are immobilized onmembranes. In both Western blotting and phage display screening,receptors are immobilized non-covalently. The receptor of interest isthen selected by its unique property, i.e., interaction with anantibody. In some other applications such as immunoprecipitation andaffinity purification, agents (e.g., antibodies, ligands) are covalentlyconjugated onto solid supports (e.g., agarose beads) through theirprimary amines, sulfhydryls or other reactive groups. In general,receptors retain their abilities of interacting with other proteins orligands after immobilization. However, even with the immobilization of amultiplicity of receptors from a sample, the problems of simultaneousdetection of receptor expression, receptor form, and receptor activityfor a multiplicity of receptors remains.

Thus, an object of the present invention is to overcome the problemsdescribed above. Accordingly, the present invention provides an approachfor the simultaneous determination of the clustering and activationstates of a plurality of receptors in single cells. This approachpermits the rapid detection of heterogeneity in a complex cellpopulation based on receptor clustering and activation states, and theidentification of cellular subsets that exhibit correlated changes inreceptor clustering and activation within the cell population. Moreover,this approach allows the correlation of cellular activities orproperties, such as surface molecule expression or cell granularity,with receptor clustering and activation at the single cell level.

SUMMARY OF THE INVENTION

In accordance with the above objects, the present invention providesmethods and compositions for simultaneously detecting the clustering andactivation state of receptor elements in single cells using flowcytometry. The invention further provides methods and compositions forsimultaneously detecting multiple clusters and activation states ofreceptor elements in single cells using flow cytometry.

In another aspect the present invention the present invention provides amethod of detecting the clustering of receptor elements in single cells,said method comprising: a) providing a sample comprising a cell, whereinthe cell comprises receptor elements; b) contacting that cell withbinding elements that bind to at least one of the receptor elements toactivate the formation of clusters, wherein each of the binding elementscomprise a label, wherein the clusters comprise; i) at least tworeceptor elements, ii) at least one binding elements bound to at leastone of the receptor elements of the cluster; and c) detecting the label.

In another embodiment the present invention provides a method ofsimultaneously detecting multiple clusters in single cells, wherein themethod comprising: a) providing a sample comprising a cell wherein thecell comprises a plurality of different receptor elements; b) contactingthe cell with a plurality of different binding elements that bind to atleast one of the different receptor elements to activate the formationof multiple clusters, wherein each of the different binding elementscomprise an identifying label, wherein the clusters comprise; i) atleast two receptor elements; ii) at least one binding element bound toat least one receptor element of the cluster; and c) detecting thelabel.

In a further embodiment the instant invention provides a method ofsimultaneously detecting the activation state and clustering of receptorelements in single cells, said method comprising: a) providing a samplecomprising a cell, wherein the cell comprises at least first receptorelements; b) contacting the cell with at least first activation-specificantibodies that binds to an isoform of the first receptor element;wherein each of first activation-specific antibodies comprise anidentifying label; c) contacting the cell with at least first bindingelements that bind to at least one of the receptor elements to activatethe formation of clusters, wherein each of the first binding elementscomprise an identifying label, wherein the clusters comprise; i) atleast two receptor elements; ii) at least one binding elements bound toat least one receptor elements of the cluster; and d) simultaneouslydetecting the identifying label of the first activation-specificantibodies and the identifying label each of the clusters.

In another embodiment, the present invention provides the methodsdescribed above, wherein the binding of the binding elements to thereceptor elements is activated by providing the cell with an activator.

In another embodiment, the present invention provides the methodsdescribed above, wherein the label of the binding element comprises anantibody conjugated to a fluorophore, and wherein the antibody binds tothe binding element.

In another embodiment, the present invention provides the methodsdescribed above, wherein the clustering activates the receptor elementsin the clusters.

In another embodiment, the present invention provides the methodsdescribed above, wherein the signal is proportional to the number of theclusters.

In another embodiment, the present invention provides the methodsdescribed above, wherein the detecting is by flow cytometry.

In another embodiment, the present invention provides the methodsdescribed above, wherein the detecting further comprises using a FACSmachine.

In another embodiment, the present invention provides the methodsdescribed above, wherein the detecting further comprises using a doubletdiscriminator.

In another embodiment, the present invention provides the methodsdescribed above, wherein the sample is from a patient.

In another embodiment, the present invention provides the methodsdescribed above wherein the receptor elements are not Leukocyte FunctionAntigen-1.

In another embodiment, the present invention provides the methodsdescribed above wherein the receptor elements are not integrins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a general diagram of receptor element clustering andactivation.

FIG. 2A-2C depict diagrams of three general mechanisms of receptorelement clustering.

FIG. 3A-3H depict results demonstrating that soluble ICAM-2 bindinginduces LFA-1 clustering and cytoskeletal polarization. FIG. 3A)Purified human ICAM-2 from retrovirally transduced NIH3T3 (see materialsand methods) was tested for purity, size, and aggregate formation bynative gel electrophoresis and gel filtration. Analysis of purifiedICAM-2 from two different preps and ICAM2-FC from NSO cells. Molecularweight was calculated using relative distance migration from a molecularweight standard. Concentration was determined using serial dilutions ofBSA as a standard. Molecular weight and concentration are displayed inthe figure. FIG. 3B) ICAM-2 ligand binding to cell surface as a functionof time. 5×10⁶ unstimulated and PMA stimulated (1 μg/ml, 30 min) JurkatT cells were incubated with ICAM-2-FITC or LFA-1-FITC antibody (1 μg/ml)and subjected to time dependent flow cytometry. Mean fluorescenceintensity was plotted per time of a gated homogenous cell population.Flow rate was maintained at 200-300 cells/second, with fluorescenceintensity values acquired at 10 millisecond intervals for 1200 seconds.FIG. 3C) Flow cytometric analysis of ICAM-2-FITC and α-LFA-1-FITCsurface binding on cytochalisin D treated (10 μM, 30 min) Jurkat cellsat 37° C. and 4° C. FIG. 3D) Curve fit analysis of ICAM-2-FITC bindingper 10⁴ cells. Jurkat cells were incubated with, ICAM-2-FITC at 37° C.for 30 min at indicated concentrations in 50 μL volume. Cells werewashed 1×, and analyzed for mean fluorescent intensity (MFI) of 10⁴cells. Percent ICAM-2-FITC bound was calculated from the followingequation:100×[(MFI_([x])-MFI_(unstained))/(MFI_([saturated])-MFI_(unstained))]where [x]=ICAM-2-FITC concentration and [saturated]=concentration thatsaturated binding. Values were fit to the equation Y=m1*M0/(m2+M0) (seematerials and methods). FIG. 3E) Native gel electrophoresis of purifiedICAM-2. Gel was coomasie stained and purified ICAM-2 did not displayaggregates (after gel filtration, we found that the added glycerol inthe purification step eliminated higher molecular weight speciesformation). FIG. 3F) Purity analysis of ICAM-2 by native PAGE. Quantitywas determined from densitometry measurements of entire lane. Percentpurity was determined from calculating density of band relative to totaldensity of the lane. ICAM-2 from prep one (left) and from prep 2(right). Purity was greater than 98%. FIG. 3G) ICAM-2 was conjugated toFITC and purified conjugate (by spin chromatography) was immunoblottedwith anti-ICAM-2 antibody (left panel) and subsequently verified forfluorescent conjugation in gel (right panel). FIG. 3H) Treatment ofcells with cytochalisin D at low concentrations at 40 C led to enhancedICAM-2 binding in a subset of cells and saturated at 370 C. Titration ofICAM-2-FITC surface binding at 370 C and 40 C in cytochalisin D treated(10 mM, 0 min) and untreated Jurkat cells by flow cytometry. MedianFluorescence Intensity (MFI) is plotted as a function of ICAM-2-FITCconcentration.

FIG. 4A-4G depict the results of experiments demonstrating thesimultaneous detection of ICAM-2 induced LFA-1 clustering, activation,and actin/microtubule reorganization. FIG. 4A) Confocal microscopy ofactin and microtubule architecture upon ICAM-2 stimulus (10 μg/ml, 30min) in Jurkat cells. Actin was stained by phalloidin-alexa633 andtubulin by taxol-alexa546. Inserts display enlarged cell image ofrepresentative treatment FIG. 4B) Flow cytometric staining for actin andmicrotubules as described above.

FIG. 4C) Left: Fluorescence topography analysis of ICAM-2-FITC (1 μg/ml,time as indicated) surface distribution. Intensity color gradientdepicts high and low fluorescent intensity values. Right: Confocalmicroscopy of ICAM-2-FITC surface binding (10 μg/ml, 30 min) andanti-β2-alexa568 (clone CTB104). FIG. 4D) Staining for ICAM-2-FITCsurface binding (fluorescence intensity) and surface distribution(fluorescence pulse) in cytochalisin D treated Jurkat cells at 37° C.and 4° C. by flow cytometry. FIG. 4E) Values for ICAM-2-FITCfluorescence intensity per time-of-flight per cell as described in text.FIG. 4F) Flow cytometric staining for LFA-1 activation bymAb24-alexa633. Stimuli as indicated (10 μM, 30 min), PMA (1 μg/ml)prior to ICAM-2 stimulation (10 μg/ml, 30 min). Staining was performedat 37° C. FIG. 4G) Mean fluorescence intensity (MFI) values of mAb24-633and ICAM-2 cluster values, computed as described above, as a function oftime.

FIG. 4H-4I depict ICAM-2 induced p44/42 MAPK phosphorylation, andinhibition by LFA-1 mAb and comparison with other stimuli. FIG. 4H)Phospho-p44/42 MAPK immunoblot of total lysates from Jurkat cellsstimulated with ICAM-2 (concentrations indicated in figure, 30 min) andsubsequently blocked with increasing concentrations of LFA-1 mAb. FIG.4I) Comparison of phospho-p44/2 MAPK induction to stimuli using FACSbased detection of phospho-p44/42 MAPK. Stimuli were either 1 mg/ml(PHA, PMA, ionomycin). 10 mg/ml (LFA-1 mAb and ICAM-2), or 10 mM forPD98059 and U0126. Chemical inhibition was done 30 min prior tostimulation. Data is presented as percent phospho-p44/24 MAPK positivecells relative to unstimulated cells. Note the difference between LFA-1mAb crosslinking and ICAM-2 stimulation.

FIG. 5A-5E depict the results of experiments demonstrating ICAM-2induced p44/42 MAPK activation via LFA-1 interaction. FIG. 5A) Meanfluorescent intensity values (MFI) of ICAM-2-FITC (adhesion) andmAb24-Alexa633 (LFA-1 activation) in treated Jurkat cells (as above).Control unstimulated and/or compound pretreated values were subtracted.FIG. 5B) Top panel: Intracellular phospho-p44/42 MAPK detection as afunction of ICAM-2 dose in Jurkat cells by flow cytometry (see Materialand Methods). Mean fluorescent intensity values (MFI) were plotted±standard deviation (SD). Bottom panel: ICAM-2 treated Jurkat cells werestained for intracellular phospho-p44/42 MAPK as described above. mAbsto β82 and α_(L) integrins (10 μg/ml, 10 minutes) were titrated prior toICAM-2 treatment as indicated and plotted for MFI±SD. FIG. 5C) ICAM-2induced p44/42 MAPK activity is blocked by β2 and α_(L) integrinantibodies as determined by a p44/42 MAPK kinase assay. Conditions forinduction and inhibition are as described above. Recombinant activep44/42 MAPK served as an internal positive control (denoted by “+”).FIG. 5D) Inhibition and activation profile for intracellularphospho-p44/42 by flow cytometry in the presences of chemical agents (10mM), EDTA (1 mM), or PMA, ionomycin and ICAM-2 stimulus (as indicatedabove). MFI values±SD are plotted. FIG. 5E) depicts that sICAM-2 inducesPyk2 and Syk membrane localization. Confocal Microscopy of Jurkat cellstreated with ICAM-2 protein (10 mg/ml) or bovine serum albumin(Unstimulated, 10 mg/ml) for 10 minutes and prepared for confocalmicroscopy (see material and methods). Cells were stained for Pyk2 andSyk. Panels A-C represents unstimulated cells and panels D-F representICAM-2 treated cells. Scale bar is denoted in lower left corner ofpanels (in micrometers).

FIG. 6A-6E depict the results of experiments demonstrating ICAM-2induced phosphorylation of Pyk2 and Syk, and β32 integrin association.FIG. 6A) Phospho-raf and phospho-p44/42 immunoblot inhibition profile bytyrosine kinase inhibitors. 1×10⁶ cells were treated with indicatedcompound (10 mM, 30 min) and then stimulated with ICAM-2 (10 mg/ml, 30min). Cell lysates were immunoblotted for phospho-raf andphospho-p44/42. Compound alone did not induce detectablephosphorylation. FIG. 6B) Pyk2 and Syk are phosphorylated andco-immunoprecipitate with b2 integrin upon ICAM-2 stimulus.Phospho-specificity was determined by phospho-PykpY402 andphospho-syk(Tyr525/526) antibodies. FIG. 6C) Kinetic analyses of thephosphorylation state of PKCa/b, Pyk2, and Syk as a function of ICAM-2stimulus per time. Cells were treated and processed as above.Phospho-specific PKCa/b_(II) (Thr638) and the following antibodies wereused; Pyk2 and Syk were first immunoprecipated, probed withanti-phosphotyrosine antibody (PY20), stripped and subsequently probedwith indicated non-phospho specific antibody. Immunoblots arerepresentative of triplicate experiments. FIG. 6D) and FIG. 6E) depictthat LFA-1 induced phosphorylation of Pyk2 and Syk is dependent on PKC.We screened for the inhibition of sICAM-2 induced Pyk2 and Sykphosphorylation by chemical inhibitors to tyrosine kinases using aphospho-tyrosine based ELISA. Pyk2 phosphorylation was abrogated in thepresence of PKC inhibitors bisindolymaleimide II (BIM II) andstaurosporine (STP), in addition to tyrphostin A9, a specific Pyk2inhibitor. Pyk2 phosphorylation was also affected by inhibitors ofphospholipase Cg (neomycin), inhibitors of Syk (piceatannol), and PKCinhibitor BIM I. Syk phosphorylation was completely abolished byinhibition of Pyk2, PLCg1, and strongly affected by PKC inhibitors.Thus, both Pyk2 and Syk phosphorylations were dependent on PKC activity,while Syk phosphorylation was additionally dependent on PLCg1 and Pyk2activity. It was not possible to assess specific PKC isozymes by thismethod. A chemical genetic approach was undertaken to determine thehierarchy of PKC, Pyk2, PLCg1, and Syk activities in response to sICAM-2stimulus by verifying phosphorylation status of each kinase in thepresence of respective chemical inhibitors. Inhibition of PKC with BIMII abrogated phosphorylation of Pyk2, PLCg1, and Syk. Inhibition ofPLCg1 by neomycin abrogated phosphorylation of Syk, with no inhibitionobserved for Pyk2. Inhibition of Syk by piceatannol did not blockphosphorylation of Pyk2 or PLCg1. These observations suggest that PKCactivation is upstream of PYK2, PLCg, and SYK activities, and also thatSYK activity is consequential to PYK2 and PLCg1 activity. Thus, theupstream signaling events from LFA-1 to Raf-1 appear to involvePKC/Pyk2/PLCg1/Syk.

FIG. 7A-7B depict the results of experiments demonstrating that ICAM-2induces cytotoxic lymphocyte activity in IL-2 activated human PBMC. FIG.7A) PBMC were either treated with ICAM-2 (10 mg/ml) in the presence ofIL-2 for 12 hr and then incubated with CFSE labeled target HL60 cells ata 50:1 E:T ratio for 4 hrs. Remaining HL60 cells were quantified by flowcytometry. FIG. 7B) PBMC were treated with IL-2 (100 U/ml, 12 hrs) andtreated with ICAM-1, -2, or 3 FC proteins (10 mg/ml) and used in acytotoxicity assay as described above. Results are representative of 4independent experiments.

FIG. 8A-1, FIG. 8A-2, FIG. 8A-3, FIG. 8A-4 and FIG. 8B-8C depict theresults of experiments demonstrating ICAM-2 exhibits differences toICAM-1 and ICAM-3 in mediating perforin and granzyme release fromCD56⁺CD8⁺ cytotoxic lymphocytes subsets. IL-2 activated PBMC (shown inFIG. 8A-1 and FIG. 8A-3) were mock-treated (IgG) (FIG. 8A-2), ICAM-1(FIG. 8A-2), ICAM-2 (FIG. 8A-4), or ICAM-3 (FIG. 8A-4) treated (10 mg/mlof FC fusion protein) for 12 hrs prior to incubation with target HL60cells at a 50:1 E:T ratio for 4 hrs. Cells were then prepared for flowcytometry with CD8-CY5PE, CD56-PE surface stains, and perforin-CY5 andgranzyme-A-FITC intracellular stains. Cells were gated forCD56⁺CD8^(low), CD56⁺CD8^(med), CD56⁺ CD8^(high), CD56⁻ CD8⁻, CD56⁻CD8^(high) populations as shown in FIG. 8A-1 and FIG. 8A-3 andpopulation frequencies within appropriate gate. FIG. 8A-2 and FIG. 8A-4are subset gated populations displayed for perforin and granzyme-A logfluorescent intensities. Results are representative of 3 independentexperiments and were similar at 25:1 and 12.5:1 E:T ratios (data notshown). Manual calibration was performed. FIG. 8B) CD56CD8 populationsubsets were gated (as indicated) and displayed for intracellularperforin for ICAM-1, -2, -3 stimulated cells. Perforin percentage wascalculated from the following equation where MFI equals mean fluorescentintensity: 100×[(MFI_(experimental)−MFI_(isotype mAb))/(MFI_(control)−MFI_(isotype mAb))].Unstimulated cells were used as control. FIG. 8C) Intracellulargranzyme-A values were calculated and displayed as described above.

FIG. 9A-9B depict the results of experiments demonstrating that ICAM-2induced LFA-1 mediated p44/42 MAPK correlates with LFA-1 activation inhuman CD56⁺CD8⁺ cells. FIG. 9A) Conjugate formation of CFSE labeled HL60cells and CD56⁺CD8⁺ cells. Conjugate flow cytometric based assay wasperformed on PBMC treated with indicated chemicals (10 mM, 30 min) priorto treatment with ICAM-2 (10 mg/ml, 30 min). CFSE labeled HL60 cellswere incubated at 25:1 E:T ratio for 5 min, and fixed with 1%paraformaldehyde. Cells were then immunolabeled with CD8 and CD56antibodies, gated for CD8⁺CD56⁺ cell populations and percent HL60fluorescence was made relative to total HL60 cells. FIG. 9B) IL-2activated PBMC were either treated with ICAM-2 (10 mg/ml, 30 min) ormock-treated (IgG) and stained for active-LFA-1 (mAb24-Alexa633),phospho-p44/42-Ax488,C08-CY5PE, and CO56-PE. CD56⁺CD8⁺ cell populationsare gated and displayed for active LFA-1 vs. phospho-p44/42. The meanfluorescent intensities (MFI) of mAb24-Ax633 and phospho-44/42-Ax488were computed and displayed over time as described above.

DETAILED DESCRIPTION OF THE INVENTION

Intracellular assays of signaling systems have been limited by aninability to correlate functional subsets of cells in complexpopulations based on the clustering and activation state of receptorelements in a cell. Such correlations are important for distinguishingchanges in signaling status that arise in rare cell subsets duringsignaling or in disease manifestations. The present invention solvesthese problems by providing methods and compositions for simultaneouslydetecting the receptor clustering state and activation state of aplurality of activatable receptor elements in single cells using flowcytometry. The invention further provides methods and compositions ofscreening for bioactive agents capable of coordinately modulating theactivity or activation state of a plurality of activatable receptorelements in single cells. The methods and compositions can be used todetermine the receptor clustering and activation profile of a cell forpredicting or diagnosing a disease state, and for monitoring treatmentof a disease state. Further, the methods and compositions of the presentinvention can be used optionally to sequentially detect the clusteringand activation state of a plurality of receptor elements in singlecells. In addition, the methods and compositions of the presentinvention can be used optionally to detect the activation state of asingle receptor element or modulate the clustering or activation stateof a single receptor element in single cells.

As shown in FIG. 1, detection of receptor element clustering may bedetermined as follows. First, a receptor element capable of associatingwith one or more receptor elements to form clusters is chosen. Next, acell type containing that receptor is chosen. The cell is then contactedwith a labeled binding element which is capable of binding to thereceptor element and initiating the formation of clustering. Clusteringof the receptor element can then be determined by detecting the labelattached to the binding element. As will be appreciated by those ofskill in the art, the labels may be any label that can be visualizedand/or measured or otherwise identified so that its presence or absencecan be determined.

Additionally, conversion of the receptor from one form to another (e.g.activation) of the receptor following the binding of a binding elementmay be detected by determining the presence of particular isoform of thereceptor. Conversion from one isoform to another either initiates asignal that mediates the effects of a signaling pathway or conversely,turns off a signal that mediates the effects of a signaling pathway.Thus, by contacting the cell with labeled antibodies specific for aparticular isoform, it is possible to determine whether or not thatisoform is present.

Accordingly, the methods and compositions of the present invention maybe used to detect clustering and conversion of receptor elements fromone isoform to another. By “receptor element” is meant any protein,membrane-bound or soluble, capable of interacting with a ligand, e.g.hormone, peptide or small molecule, resulting in the propagation of anintra- or extracellular signal. The term receptor element, as usedherein, encompasses both naturally occurring, mutant and recombinantreceptors.

In preferred embodiment, the receptor element is capable of clustering.By “clustering”, and grammatical equivalents used herein, is meant anyreversible or irreversible association of one or more receptor elements.Clusters can be made up of 2, 3, 4, etc., receptor elements. Clusters oftwo elements are termed dimers. Clusters of 3 or more receptor elementsare generally termed oligomers, with individual numbers of clustershaving their own designation, for example, a cluster of 3 receptorelements is a trimer, a cluster of 4 receptor elements is a tetramer,etc.

Clusters can be made up of identical receptor elements or differentreceptor elements. Clusters of identical receptor elements are termed“homo” clusters, while clusters of different receptor elements aretermed “hetero” clusters. Accordingly, a cluster can be a homodimer, asis the case for the β₂-adrenergic receptor. Alternatively, a cluster canbe a heterodimer, as is the case for GABA_(B)-R. In other embodiments,the cluster is a homotrimer, as in the case of TNFα, or a heterotrimersuch the one formed by membrane-bound and soluble CD95 to modulateapoptosis. In further embodiments the cluster is a homo-oligomer, as inthe case of Thyrotropin releasing hormone receptor, or ahetero-oligomer, as in the case of TGFβ1.

Activation or conversion of a receptor element from one isoform toanother may be mediated either by receptor clustering or by the bindingof a binding element to the receptor. Generally, initiation ofclustering via the binding of a binding element results in theactivation or signaling potential of a receptor. As will be appreciatedby those of skill in the art, activation encompasses both the “turningon” or “turning”off” of a signal used to mediate a signaling pathway.

In a preferred embodiment, the activation or signaling potential ofreceptor elements is mediated by clustering, irrespective of the actualmechanism by which the receptor clustering is induced. As shown in FIG.2, clustering can occur in a variety of ways. The definition includesreceptors that are normally activated to cluster a) as membrane boundreceptors by binding to ligands, b) as membrane bound receptors bybinding to other surface molecules, or c) as intracellular (non-membranebound) receptors binding to ligands.

In a preferred embodiment membrane bound receptor elements cluster uponligand binding. One class of receptor elements includes membrane boundproteins, or complexes of proteins, which are activated to cluster uponligand binding. As is known in the art, these receptor elements can havea variety of forms, but in general they comprise at least three domains.First, these receptors have a ligand binding domain, which can beoriented either extracellularly or intracellularly. Next, thesereceptors have a memebrane-binding domain, which can take the form of aseven pass transmembrane domain (discussed below in connection withG-protein-coupled receptors) or a lipid modification, such asmyristylation, to one of the receptor's amino acids which allows formembrane association when the lipid inserts itself into the lipidbilayer. Finally, the receptor has an signaling domain, which isresponsible for propagating the downstream effects of the receptor.

Examples of such receptor elements include hormone receptors, cytokinereceptors, steroid receptors, adhesion receptors, PDGF (platelet derivedgrowth factor receptor), EGF (epidermal growth factor), VEGF (vascularendothelial growth factor), uPAR (urokinase plasminogen activatorreceptor), ACHR (acetylcholine receptor), IgE (immunoglobulin Ereceptor), estrogen receptor, thyroid hormone receptor, integrins (β1,β2, β3, β4, β5, β6, α1, α2, α3, α4, α5, α6), MAC-1 (β2 and cd11b), αVβ3,opiod receptors (mu and kappa), FC receptors, serotonin receptors (5-HT,5-HT6, 5-HT7), β-adrenergic receptors, insulin receptor, leptinreceptor, TNF receptor (tissue-necrosis factor), cytokine and chemokinereceptors (IL1-a, IL-b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10. IL-12, IL-15, IL-18, IL-21, CCR5, CCR7, CXCR4, CCR-1-10, CCL20),statin receptors, FAS receptor, BAFF receptor, FLT3 receptor, GMCSFreceptor, and fibronectin receptor.

In a preferred embodiment the receptor element is a cytokine receptor.Cytokines are a family of soluble mediators of cell-to-cellcommunication that includes interleukins, interferons, andcolony-stimulating factors. The characteristic features of cytokines liein their functional redundancy and pleiotropy. Most of the cytokinereceptors that constitute distinct superfamilies do not possessintrinsic protein tyrosine kinase domains, yet receptor stimulationusually invokes rapid tyrosine phosphorylation of intracellularproteins, including the receptors themselves. Many members of thecytokine receptor superfamily activate the Jak protein tyrosine kinasefamily, with resultant phosphorylation of the STAT transcriptionalactivator factors. IL-2, 1L-4, IL-7 and Interferon y have all been shownto activate Jak kinases (Frank et al. (1995) Proc. Natl. Acad. Sci. USA92:7779-7783); Scharfe et al. (1995) Blood 86:2077-2085); (Bacon et al.(1995) Proc. Natl. Acad. Sci. USA 92:7307-7311); and (Sakatsume et al.(1995) J. Biol. Chem. 270:17528-17534). Events downstream of Jakphosphorylation have also been elucidated. For example, exposure of Tlymphocytes to IL-2 has been shown to lead to the phosphorylation ofsignal transducers and activators of transcription (STAT) proteinsSTAT1α, STAT1β, and STAT3, as well as of two STAT-related proteins, p94and p95. The STAT proteins were found to translocate to the nucleus andto bind to a specific DNA sequence, thus suggesting a mechanism by whichIL-2 may activate specific genes involved in immune cell function (Franket al. supra). Jak3 is associated with the gamma chain of the IL-2,IL-4, and IL-7 cytokine receptors (Fujii et al. (1995) Proc. Natl. Acad.Sci. 92:5482-5486) and (Musso et al. (1995) J. Exp. Med. 181:1425-1431).The Jak kinases have also been shown to be activated by numerous ligandsthat signal via cytokine receptors such as, growth hormone anderythropoietin and IL-6 (Kishimoto (1994) Stem cells Suppl. 12:37-44).

In a preferred embodiment the receptor element is a member of the nervegrowth factor receptor superfamily, such as the Tumor necrosis factor areceptor. Tumor necrosis factor a (TNF-α or TNF-alpha) is a pleiotropiccytokine that is primarily produced by activated macrophages andlymphocytes; but is also expressed in endothelial cells and other celltypes. TNF-alpha is a major mediator of inflammatory, immunological, andpathophysiological reactions. (Grell, M., et al., (1995) Cell,83:793-802). Two distinct forms of TNF exist, a 26 kDa membraneexpressed form and the soluble 17 kDa cytokine which is derived fromproteolytic cleavage of the 26 kDa form. The soluble TNF polypeptide is157 amino acids long and is the primary biologically active molecule.

TNF-alpha exerts its biological effects through interaction withhigh-affinity cell surface receptors. Two distinct membrane TNF-alphareceptors have been cloned and characterized. These are a 55 kDaspecies, designated p55 TNF-R and a 75 kDa species designated p75 TNF-R(Corcoran. A. E., et al., (1994) Eur. J. Biochem., 223:831-840). The twoTNF receptors exhibit 28% similarity at the amino acid level. This isconfined to the extracellular domain and consists of four repeatingcysteine-rich motifs, each of approximately 40 amino acids. Each motifcontains four to six cysteines in conserved positions. Dayhoff analysisshows the greatest intersubunit similarity among the first three repeatsin each receptor. This characteristic structure is shared with a numberof other receptors and cell surface molecules, which comprise theTNF-R/nerve growth factor receptor superfamily (Corcoran. A. E., et al.,(1994) Eur. J. Biochem., 223:831-840).

TNF signaling is initiated by receptor clustering, either by thetrivalent ligand TNF or by cross-linking monoclonal antibodies(Vandevoorde, V., et al., (1997) J. Cell Biol., 137:1627-1638).Crystallographic studies of TNF and the structurally related cytokine,lymphotoxin (LT) have shown that both cytokines exist as homotrimers,with subunits packed edge to edge in a threefold symmetry. Structurally,neither TNF or LT reflect the repeating pattern of the their receptors.Each monomer is cone shaped and contains two hydrophilic loops onopposite sides of the base of the cone. Recent crystal structuredetermination of a p55 soluble TNF-R/LT complex has confirmed thehypothesis that loops from adjacent monomers join together to form agroove between monomers and that TNF-R binds in these grooves (Corcoran.A. E., et al., (1994) Eur. J. Biochem., 223:831-840).

In preferred embodiment, the receptor element is a receptor tyrosinekinase. The receptor tyrosine kinases can be divided into five subgroupson the basis of structural similarities in their extracellular domainsand the organization of the tyrosine kinase catalytic region in theircytoplasmic domains. Sub-groups I (epidermal growth factor (EGF)receptor-like), II (insulin receptor-like) and the EPH/ECK familycontain cysteine-rich sequences (Hirai et al., (1987) Science238:1717-1720 and Lindberg and Hunter, (1990) Mol. Cell. Biol.10:6316-6324). The functional domains of the kinase region of thesethree classes of receptor tyrosine kinases are encoded as a contiguoussequence (Hanks et al. (1988) Science 241:42-52). Subgroups III(platelet-derived growth factor (PDGF) receptor-like) and IV (thefibro-blast growth factor (FGF) receptors) are characterized as havingimmunoglobulin (Ig)-like folds in their extracellular domains, as wellas having their kinase domains divided in two parts by a variablestretch of unrelated amino acids (Yanden and Ullrich (1988) supra andHanks et al. (1988) supra).

The family with by far the largest number of known members is the EPHfamily. Since the description of the prototype, the EPH receptor (Hiraiet al. (1987) Science 238:1717-1720), sequences have been reported forat least ten members of this family, not counting apparently orthologousreceptors found in more than one species. Additional partial sequences,and the rate at which new members are still being reported, suggest thefamily is even larger (Maisonpierre et al. (1993) Oncogene 8:3277-3288;Andres et al. (1994) Oncogene 9:1461-1467; Henkemeyer et al. (1994)Oncogene 9:1001-1014; Ruiz et al. (1994) Mech. Dev. 46:87-100; Xu et al.(1994) Development 120:287-299; Zhou et al. (1994) J. Neurosci. Res.37:129-143; and references in Tuzi and Gullick (1994) Br. J. Cancer69:417-421). Remarkably, despite the large number of members in the EPHfamily, all of these molecules were identified as orphan receptorswithout known ligands.

As used herein, the terms “EPH receptor” or “EPH-type receptor” refer toa class of receptor tyrosine kinases, comprising at least elevenparalogous genes, though many more orthologs exist within this class,e.g. homologs from different species. EPH receptors, in general, are adiscrete group of receptors related by homology. and easilyrecognizable, e.g., they are typically characterized by an extracellulardomain containing a characteristic spacing of cysteine residues near theN-terminus and two fibronectin type III repeats (Hirai et al. (1987)Science 238:1717-1720; Lindberg et al., (1990) Mol. Cell Biol.10:6316-6324; Chan et al. (1991) Oncogene 6:1057-1061; Maisonpierre etal. (1993) Oncogene 8:3277-3288; Andres et al. (1994) Oncogene9:1461-1467; Henkemeyer et al. (1994) Oncogene 9:1001-1014; Ruiz et al.(1994) Mech. Dev. 46:87-100; Xu et al. (1994) Development 120:287-299;Zhou et al. (1994) J. Neurosci. Res. 37:129-143; and references in Tuziand Gullick (1994) Br. J. Cancer 69:417-421). Exemplary EPH receptorsinclude the eph, elk, eck, sek, mek4, hek, hek2, eek, erk, tyro1, tyro4,tyro5, tyro6, tyro11, cek4, cek5, cek6, cek7, cek8, cek9, cek10, bsk,rtk1, rtk2, rtk3, myk1, myk2, ehkl, ehk2, pagliaccio, htk, erk and nukreceptors.

In another embodiment the receptor element is a member of thehematopoietin receptor superfamily. Hematopoietin receptor superfamilyis used herein to define single-pass transmembrane receptors, with athree-domain architecture: an extracellular domain that binds theactivating ligand, a short transmembrane segment, and a domain residingin the cytoplasm. The extracellular domains of these receptors have lowbut significant homology within their extracellular ligand-bindingdomain comprising about 200-210 amino acids. The homologous region ischaracterized by four cysteine residues located in the N-terminal halfof the region, and a Trp-Ser-X-Trp-Ser (WSXWS) motif located justoutside the membrane-spanning domain. Further structural and functionaldetails of these receptors are provided by Cosman, D. et al., (1990).The receptors of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, prolactin,placental lactogen, growth hormone GM-CSF, G-CSF, M-CSF anderythropoietin have, for example, been identified as members of thisreceptor family.

In a further embodiment, the receptor element is an integrin other thanLeukocyte Function Antigen-1 (LFA-1). Members of the integrin family ofreceptors function as heterodimers, composed of various α and βsubunits, and mediate interactions between a cell's cytoskeleton and theextracellular matrix. (Reviewed in, Giancotti and Ruoslahti, Science285, 13 Aug. 1999). Different combinations of the α and β subunits giverise to a wide range of ligand specificities, which may be increasedfurther by the presence of cell-type-specific factors. Integrinclustering is know to activate a number of intracellular signals, suchas RAS, MAP kinase, and phosphotidylinosital-3-kinase. In a preferredembodiment the receptor element is a heterodimer (other than LFA-1)composed of a 6 integrin and an a integrin chosen from the followingintegrins; β1, β2, β3, β4, β5, β6, α1, α2, α3, α4, α5, and α6, or isMAC-1 (β2 and cd11b), or αVβ3.

In a preferred embodiment, the receptor element is not the integrinLFA-1.

In an alternative embodiment, the receptor element is not an integrin.

In another embodiment the receptor elements cluster for signaling bycontact with other surface molecules. In contrast to the receptorsdiscussed above, these receptor elements cluster for signaling bycontact with other surface molecules, and generally use moleculespresented on the surface of a second cell as ligands. Receptors of thisclass are important in cell-cell interactions, such mediating cell tocell adhesion and immunorecognition.

Examples of such receptor elements are CD3 (T cell receptor complex),BCR (B cell receptor complex), CD4, CD28, CD80, CD86, CD54, CD102, CD50and ICAMs 1, 2 and 3.

In a preferred embodiment the receptor element is a T cell receptorcomplex (TCR). TCRs occur as either of two distinct heterodimers, αβ orγδ, both of which are expressed with the non polymorphic CD3polypeptides γ; δ, ε, ζ. The CD3 polypeptides, especially ζ and itsvariants, are critical for intracellular signaling. The αβ TCRheterodimer expressing cells predominate in most lymphoid compartmentsand are responsible for the classical helper or cytotoxic T cellresponses. Im most cases, the αβ TCR ligand is a peptide antigen boundto a class I or a class II MHC molecule (Fundamental Immunology, fourthedition, W. E. Paul, ed., Lippincott-Raven Publishers, 1999, Chapter 10,pp 341-367).

In a preferred embodiment the receptor element is a B cell antigenreceptor (BCR). Antigen contact with a specific B cell triggers thetransmembrane signaling function of the BCR. BCR molecules are rapidlyinternalized after antigen binding, leading to antigen uptake anddegradation in endosomes or lysosomes. In the case of protein antigens,antigen-derived peptides bind in the groove of class II MHC molecules.Upon binding, this complex is sent to the cell surface, where it servesas a stimulus for specific helper T cells. Antigen recognition by thehelper T cell induces it to form a tight and long lasting interactionwith the B cell and to synthesize B cell growth and differentiationfactors. B cells activated in this way may proliferate and terminallydifferentiate to antibody secreting cells (also called plasma cells)(Fundamental Immunology, fourth edition, W. E. Paul, ed.,Lippincott-Raven Publishers, 1999, Chapters 6-7, pp 183-261)

In a preferred embodiment the receptor element is an intracellularadhesion molecule (ICAM). ICAMs-1, -2, and -3 are cellular adhesionmolecules belonging to the immunogloblin superfamily. Each of thesereceptors has a single membrane-spanning domain and all bind to β2integrins via extracellular binding domains similar in structure toIg-loops. (Signal Transduction, Gomperts, et al., eds, Academic PressPublishers, 2002, Chapter 14, pp 318-319).

In a preferred embodiment the receptor elements are intracellularreceptors capable of clustering. Receptors of this class are notmembrane-bound, rather they are free to diffuse through theintracellular matrix where they bind soluble ligands prior to clusteringand signal transduction. In contrast to the previously describedreceptors, many members of this class are capable of binding DNA afterclustering to directly effect changes in RNA transcription.

In a preferred embodiment the intracellular receptors capable ofclustering are perioxisome proliferator-activated receptors (PPAR).PPARs are soluble receptors responsive to lipophillic compounds, andinduce various genes involved in fatty acid metabolism. The three PPARsubtypes, PPAR a, 3,and y have been shown to bind to DNA after ligandbinding and heterodimerization with retinoid X receptor. (Summanasekera,et al., J Biol Chem, M211261200, Dec. 13, 2002.)

In another embodiment, the receptor element is a member of the largefamily of G-protein-coupled receptors. It has recently been reportedthat a G-protein-coupled receptors are capable of clustering. (Kroeger,et al., J Biol Chem 276:16, 12736-12743, Apr. 20, 2001; Bai, et al., JBiol Chem 273:36, 23605-23610, Sep. 4, 1998; Rocheville, et al., J BiolChem 275 (11), 7862-7869, Mar. 17, 2000). As used hereinG-protein-coupled receptor, and grammatical equivalents thereof, refersto the family of receptors that bind to heterotrimeric “G proteins.”Many different G proteins are known to interact with receptors. Gprotein signaling systems include three components: the receptor itself,a GTP-binding protein (G protein), and an intracellular target protein.The cell membrane acts as a switchboard. Messages arriving throughdifferent receptors can produce a single effect if the receptors act onthe same type of G protein. On the other hand, signals activating asingle receptor can produce more than one effect if the receptor acts ondifferent kinds of G proteins, or if the G proteins can act on differenteffectors.

In their resting state, the G proteins, which consist of alpha (α), beta(β) and gamma (γ) subunits, are complexed with the nucleotide guanosinediphosphate (GDP) and are in contact with receptors. When a hormone orother first messenger binds to a receptor, the receptor changesconformation and this alters its interaction with the G protein. Thisspurs the a subunit to release GDP, and the more abundant nucleotideguanosine triphophate (GTP), replaces it, activating the G protein. TheG protein then dissociates to separate the a subunit from the stillcomplexed beta and gamma subunits. Either the Gα subunit, or the Gβγcomplex, depending on the pathway, interacts with an effector. Theeffector (which is often an enzyme) in turn converts an inactiveprecursor molecule into an active “second messenger,” which may diffusethrough the cytoplasm, triggering a metabolic cascade. After a fewseconds, the Gα converts the GTP to GDP, thereby inactivating itself.The inactivated Gα may then reassociate with the GGβγ complex.

Hundreds, if not thousands, of receptors convey messages throughheterotrimeric G proteins, of which at least 17 distinct forms have beenisolated. Although the greatest variability has been seen in the asubunit, several different β and γ structures have been reported. Thereare, additionally, many different G protein-dependent effectors.

Most G protein-coupled receptors are comprised of a single protein chainthat is threaded through the plasma membrane seven times. Such receptorsare often referred to as seven-transmembrane receptors (STRs). More thana hundred different STRs have been found, including many distinctreceptors that bind the same ligand, and there are likely many more STRsawaiting discovery.

In addition, STRs have been identified for which the natural ligands areunknown; these receptors are termed “orphan” G protein-coupledreceptors, as described above. Examples include receptors cloned byNeote et al. (1993) Cell 72, 415; Kouba et al. FEBS Lett. (1993) 321,173; and Birkenbach et al. (1993) J. Virol. 67, 2209.

Known ligands for G protein coupled receptors include: purines andnucleotides, such as adenosine, cAMP, ATP, UTP, ADP, melatonin and thelike; biogenic amines (and related natural ligands), such as5-hydroxytryptamine, acetylcholine, dopamine, adrenaline, histamine,noradrenaline, tyramine/octopamine and other related compounds; peptidessuch as adrenocorticotrophic hormone (acth), melanocyte stimulatinghormone (msh), melanocortins, neurotensin (nt), bombesin and relatedpeptides, endothelins, cholecystokinin, gastrin, neurokinin b (nk3),invertebrate tachykinin-like peptides, substance k (nk2), substance p(nk1), neuropeptide y (npy), thyrotropin releasing-factor (trf),bradykinin, angiotensin ii, beta-endorphin, c5a anaphalatoxin,calcitonin, chemokines (also called intercrines), corticotrophicreleasing factor (crf), dynorphin, endorphin, fmlp and other formylatedpeptides, follitropin (fsh), fungal mating pheromones, galanin, gastricinhibitory polypeptide receptor (gip), glucagon-like peptides (glps),glucagon, gonadotropin releasing hormone (gnrh), growth hormonereleasing hormone(ghrh), insect diuretic hormone, interleukin-8,leutropin (1 h/hcg), met-enkephalin, opioid peptides, oxytocin,parathyroid hormone (pth) and pthrp, pituitary adenylyl cyclaseactivating peptide (pacap), secretin, somatostatin, thrombin,thyrotropin (tsh), vasoactive intestinal peptide (vip), vasopressin,vasotocin; eicosanoids such as ip-prostacyclin, pg-prostaglandins,tx-thromboxanes; retinal based compounds such as vertebrate 11-cisretinal, invertebrate 11-cis retinal and other related compounds; lipidsand lipid-based compounds such as cannabinoids, anandamide,lysophosphatidic acid, platelet activating factor, leukotrienes and thelike; excitatory amino acids and ions such as calcium ions andglutamate.

Preferred G protein coupled receptors include, but are not limited to:α1-adrenergic receptor, α1B-adrenergic receptor, α2-adrenergic receptor,α2B-adrenergic receptor, β1-adrenergic receptor, β2-adrenergic receptor,β3-adrenergic receptor, m1 acetylcholine receptor (AChR), m2 AChR, m3AChR, m4 AChR, m5 AChR, D1 dopamine receptor, D2 dopamine receptor, D3dopamine receptor, D4 dopamine receptor, D5 dopamine receptor, A1adenosine receptor, Ata adenosine receptor, A2b adenosine receptor, A3adenosine receptor, 5-HT1a receptor, 5-HT1b receptor, 5HT1-likereceptor, 5-HT1d receptor, 5HT1d-like receptor, 5HT1d beta receptor,substance K (neurokinin A) receptor, fMLP receptor (FPR), fMLP-likereceptor (FPRL-1), angiotensin II type 1 receptor, endothelin ETAreceptor, endothelin ETB receptor, thrombin receptor, growthhormone-releasing hormone (GHRH) receptor, vasoactive intestinal peptidereceptor, oxytocin receptor, somatostatin SSTR1 and SSTR2, SSTR3,cannabinoid receptor, follicle stimulating hormone (FSH) receptor,leutropin (LH/HCG) receptor, thyroid stimulating hormone (TSH) receptor,thromboxane A2 receptor, platelet-activating factor (PAF) receptor,

C5a anaphylatoxin receptor, CXCR1 (IL-8 receptor A), CXCR2 (IL-8receptor B), Delta Opioid receptor, Kappa Opioid receptor,mip-1alpha/RANTES receptor (CRR1), Rhodopsin, Red opsin, Green opsin,Blue opsin, metabotropic glutamate mGluR1-6, histamine H2 receptor, ATPreceptor, neuropeptide Y receptor, amyloid protein precursor receptor,insulin-like growth factor II receptor, bradykinin receptor,gonadotropin-releasing hormone receptor, cholecystokinin receptor,melanocyte stimulating hormone receptor, antidiuretic hormone receptor,glucagon receptor, and adrenocorticotropic hormone II receptor. Inaddition, there are at least five receptors (CC and CXC receptors)involved in HIV viral attachment to cells. The two major co-receptorsfor HIV are CXCR4, (fusin receptor, LESTR, SDF1 receptor) and CCR5(m-trophic). More preferred receptors include the following humanreceptors: melatonin receptor 1a, galanin receptor 1, neurotensinreceptor, adenosine receptor 2a, somatostatin receptor 2 andcorticotropin releasing factor receptor 1. Melatonin receptor 1a isparticularly preferred. Other G protein coupled receptors (GPCRs) areknown in the art.

The methods and compositions further provide for activating receptorelements by contacting the cell comprising the receptor elements with abinding element. As used herein, the term activating and grammaticalequivalents thereof, is meant to include a step that induces a receptorelement to become active. By active herein is meant a change in theisoform of the receptor resulting in either the turning on or turningoff of a signal involved in a signaling pathway. A receptor element maybe induced to become active either through: 1) the formation of clustersinitiated by the binding of a binding element; or 2) directly throughthe binding of a binding element. For example, this step may take theform of administration of an extracellular signal, such as a smallmolecule, a change in temperature, or other alteration of theextracellular environment. In addition, this step may originateintracellularly and take the form of a change in transcription ortranslation rates.

In a preferred embodiment activation is achieved by the administrationbinding element, which can take the form of an extracellular signal. Theterm extracellular signal is intended to encompass molecules and changesin the environment that are transduced intracellularly via cell surfaceproteins that interact, directly or indirectly, with the extracellularsignal. An extracellular signal or effector molecule includes anycompound or substance that in some manner alters the activity of a cellsurface protein. Examples of such signals include, but are not limitedto, molecules such as acetylcholine, growth factors and hormones,lipids, sugars and nucleotides that bind to cell surface and/orintracellular receptors and ion channels and modulate the activity ofsuch receptors and channels. The term, “extracellular signal” alsoincludes as yet unidentified substances that modulate the activity of acellular receptor, and thereby influence intracellular functions. Suchextracellular signals are potential pharmacological agents that may beused to treat specific diseases by modulating the activity of specificcell surface receptors.

As used herein, “cell surface receptor” refers to molecules that occuron the surface of cells, interact with the extracellular environment,and transmit or transduce the information regarding the environmentintracellularly in a manner that may modulate intracellular secondmessenger activities or transcription of specific promoters, resultingin transcription of specific genes.

In a preferred embodiment, the receptor element is activated topropagate a signal by clustering. In this embodiment, the bindingelement merely activates clustering of the receptor elements, whileintermolecular interactions between the clustered receptor elements,e.g. the creation of disulfide bridges, ionic and polar interactions,etc., activates downstream signaling by the receptor. For example,receptor clustering can induce structural changes that result in thereceptor gaining kinase or phosphotase acitivity.

By “binding element”, and grammatical equivalents thereof, is meant anymolecule, e.g., protein, small organic molecule, carbohydrates(including polysaccharides), polynucleotide, lipids, etc., as describedbelow. Examples of binding elements are, transcription factors, mRNAs,tRNAs, cell surface receptors, proteinaceous and nonproteinaceoushormones as well as phospholipids, and sugars such as glucose andglycogen.

In a preferred embodiment binding elements are themselves activatible.As discussed in connection with activatible receptor elements, thebinding element activator can take many forms, e.g. small molecules,proteins, nucleic acids, changes in extra- or intracellular conditions,etc.

In one embodiment, the BE is a protein, as used herein, the terms“protein” and “polypeptide” may be used interchangeably and mean atleast two covalently attached amino acids, which includes proteins,polypeptides, oligopeptides and peptides. The protein may be made up ofnaturally occurring amino acids and peptide bonds, or syntheticpeptidomimetic structures. Thus “amino acid”, or “peptide residue”, asused herein means both naturally occurring and synthetic amino acids.For example, homo-phenylalanine, citrulline and noreleucine areconsidered amino acids for the purposes of the invention. “Amino acid”also includes imino acid residues such as proline and hydroxyproline.The side chains may be in either the (R) or the (S) configuration. Inthe preferred embodiment, the amino acids are in the (S) orL-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradation. Proteins including non-naturally occurringamino acids may be synthesized or in some cases, made recombinantly; seevan Hest et al., FEBS Lett 428:(1-2) 68-70 May 22 1998 and Tang et al.,Abstr. Pap Am. Chem. S218: U138 Part 2 Aug. 22, 1999, both of which areexpressly incorporated by reference herein.

Aromatic amino acids may be replaced with D- or L-naphylalanine, D- orL-phenylglycine, D- or L-2-thieneylalanine, D- or L-1-, 2-, 3- or4-pyreneylalanine, D- or L-3-thieneylalanine, D- orL-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- orL-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine,D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine,D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- orL-p-methoxybiphenylphenylalanine, D- or L-2-indole(alkyl)alanines, andD- or L-alkylalanines where alkyl may be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, and non-acidic amino acids of C1-C20.

Acidic amino acids can be substituted with non-carboxylate amino acidswhile maintaining a negative charge, and derivatives or analogs thereof,such as the non-limiting examples of (phosphono)alanine, glycine,leucine, isoleucine, threonine, or serine; or sulfated (e.g., —SO3H)threonine, serine, or tyrosine.

Other substitutions may include nonnatural hydroxylated amino acids maymade by combining “alkyl” with any natural amino acid. The term “alkyl”as used herein refers to a branched or unbranched saturated hydrocarbongroup of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl,isoptopyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl,hexadecyl; eicosyl, tetracisyl and the like. Alkyl includes heteroalkyl,with atoms of nitrogen, oxygen and sulfur. Preferred alkyl groups hereincontain 1 to 12 carbon atoms. Basic amino acids may be substituted withalkyl groups at any position of the naturally occurring amino acidslysine, arginine, ornithine, citrulline, or (guanidino)-acetic acid, orother (guanidino)alkyl-acetic acids, where “alkyl” is define as above.Nitrile derivatives (e.g., containing the CN-moiety-in place of COOH)may also be substituted for asparagine or glutamine, and methioninesulfoxide may be substituted for methionine. Methods of preparation ofsuch peptide derivatives are well known to one skilled in the art.

In addition, any amide linkage in any of the variant polypeptides may bereplaced by a ketomethylene moiety. Such derivatives are expected tohave the property of increased stability to degradation by enzymes, andtherefore possess advantages for the formulation of compounds which mayhave increased in vivo half lives, as administered by oral, intravenous,intramuscular, intraperitoneal, topical, rectal, intraocular, or otherroutes.

Additional amino acid modifications of amino acids of variantpolypeptides of to the present invention may include the following:Cysteinyl residues may be reacted with alpha-haloacetates (andcorresponding amines), such as 2-chloroacetic acid or chloroacetamide,to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinylresidues may also be derivatized by reaction with compounds such asbromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues may be derivatized by reaction with compounds such asdiethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain, and para-bromophenacyl bromide mayalso be used; e.g., where the reaction is preferably performed in 0.1Msodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues may be reacted with compounds suchas succinic or other carboxylic acid anhydrides. Derivatization withthese agents is expected to have the effect of reversing the charge ofthe lysinyl residues.

Other suitable reagents for derivatizing alpha-amino-containing residuesinclude compounds such as imidoesters, e.g., as methyl picolinimidate;pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate. Arginyl residues may bemodified by reaction with one or several conventional reagents, amongthem phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrinaccording to known method steps. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group. The specific modification of tyrosyl residues perse is well known, such as for introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane.

N-acetylimidizol and tetranitromethane may be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Carboxyl sidegroups (aspartyl or glutamyl) may be selectively modified by reactionwith carbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermoreaspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues may be deamidated under mildly acidic conditions. Either formof these residues falls within the scope of the present invention.

In an additional embodiment the BE is a nucleic acid. By “nucleic acid”or “oligonucleotide” or grammatical equivalents herein means at leasttwo nucleotides covalently linked together. A nucleic acid of thepresent invention will generally contain phosphodiester bonds, althoughin some cases, as outlined below, nucleic acid analogs are included thatmay have alternate backbones, comprising, for example, phosphoramide(Beaucage et al., Tetrahedron 49 (10):1925 (1993) and referencestherein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur.J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487(1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am.Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:14191986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of additional moieties such as labels, or to increase thestability and half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. As used herein, the term“nucleoside” includes nucleotides and nucleoside and nucleotide analogs,and modified nucleosides such as amino modified nucleosides. Inaddition, “nucleoside” includes non-naturally occurring analogstructures. Thus for example the individual units of a peptide nucleicacid, each containing a base, are referred to herein as a nucleoside.

As described above generally for proteins, nucleic acid BEs may benaturally occurring nucleic acids, random nucleic acids, or “biased”random nucleic acids. For example, digests of prokaryotic or eukaryoticgenomes may be used as is outlined above for proteins. Where theultimate expression product is a nucleic acid, at least 10, preferablyat least 12, more preferably at least 15, most preferably at least 21nucleotide positions need to be randomized, with more preferable if therandomization is less than perfect. Similarly, at least 5, preferably atleast 6, more preferably at least 7 amino acid positions need to berandomized; again, more are preferable if the randomization is less thanperfect.

In a preferred embodiment, the BE is a mutant cDNA encoding acatalytically inactive polypeptide. Examples of such catalyticallyinactive polypeptides include, but are not limited to, catalyticallyinactive activatable proteins and, more specifically, catalyticallyinactive kinases (e.g., PI3K) or caspases.

In a preferred embodiment, the BE is an RNA, for example an antisenseRNA or siRNA (small inhibitory RNA). In another preferred embodiment,the siRNA cleaves RNA encoding an activatable protein. The siRNAs can beprepared using the methods described herein and known in the art.

In a preferred embodiment, the BEs are synthetic compounds. Any numberof techniques are available for the random and directed synthesis of awide variety of organic compounds and biomolecules, including expressionof randomized oligonucleotides. See for example WO 94/24314, herebyexpressly incorporated by reference, which discusses methods forgenerating new compounds, including random chemistry methods as well asenzymatic methods.

Alternatively, a preferred embodiment utilizes natural compounds, asBEs, in the form of bacterial, fungal, plant and animal extracts thatare available or readily produced.

Additionally, natural or synthetically produced compounds are readilymodified through conventional chemical, physical and biochemical means.Known pharmacological agents may be subjected to directed or randomchemical modifications, including enzymatic modifications, to produceBEs that may be used in the instant invention.

In another preferred embodiment the BE is a small organic compound. BEscan synthesized from a series of substrates that can be chemicallymodified. “Chemically modified” herein includes traditional chemicalreactions as well as enzymatic reactions. These substrates generallyinclude, but are not limited to, alkyl groups (including alkanes,alkenes, alkynes and heteroalkyl), aryl groups (including arenes andheteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids,esters, amides, cyclic compounds, heterocyclic compounds (includingpurines, pyrimidines, benzodiazepins, beta-lactams, tetracylines,cephalosporins, and carbohydrates), steroids (including estrogens,androgens, cortisone, ecodysone, etc.), alkaloids (including ergots,vinca, curare, pyrollizdine, and mitomycines), organometallic compounds,hetero-atom bearing compounds, amino acids, and nucleosides. Chemical(including enzymatic) reactions may be done on the moieties to form newsubstrates or BEs which can then be used in the present invention.

In a preferred embodiment the BE is a carbohydrate. As used herein theterm carbohydrate is meant to include any compound with the generalformula (CH₂)_(n). Examples of preferred carbohydrates are di-, tri- andoligosaccharides, as well polysaccharides such as glycogen, cellulose,and starches.

In a preferred embodiment the BE is a lipid. As used herein the termlipid herein is meant to include any water insoluble organic moleculethat is soluble in nonpolar organic solvents. Examples of preferredlipids are steroids, such as cholesterol, and phospholipids such assphingomeylin.

As discussed above, the instant invention provides methods andcompositions for the detection of the clustering and activation ofreceptor elements in cells. As used herein the term cells andgrammatical equivalents herein in meant any cell, preferably anyprokaryotic or eukaryotic cell.

Suitable prokaryotic cells include, but are not limited to, bacteriasuch as E. coli, various Bacillus species, and the extremophile bacteriasuch as thermophiles, etc.

Suitable eukaryotic cells include, but are not limited to, fungi such asyeast and filamentous fungi, including species of Aspergillus,Trichoderma, and Neurospora; plant cells including those of corn,sorghum, tobacco, canola, soybean, cotton, tomato, potato, alfalfa,sunflower, etc.; and animal cells, including fish, birds and mammals.Suitable fish cells include, but are not limited to, those from speciesof salmon, trout, tulapia, tuna, carp, flounder, halibut, swordfish, codand zebrafish. Suitable bird cells include, but are not limited to,those of chickens, ducks, quail, pheasants and turkeys, and other junglefoul or game birds. Suitable mammalian cells include, but are notlimited to, cells from horses, cows, buffalo, deer, sheep, rabbits,rodents such as mice, rats, hamsters and guinea pigs, goats, pigs,primates, marine mammals including dolphins and whales, as well as celllines, such as human cell lines of any tissue or stem cell type, andstem cells, including pluripotent and non-pluripotent, and non-humanzygotes.

Suitable cells also include cell types implicated in a wide variety ofdisease conditions are particularly. Accordingly, suitable eukaryoticcell types include, but are not limited to, tumor cells of all types(particularly melanoma, myeloid leukemia, carcinomas of the lung,breast, ovaries, colon, kidney, prostate, pancreas and testes),cardiomyocytes, endothelial cells. epithelial cells, lymphocytes (T-celland B cell), mast cells, eosinophils, vascular intimal cells,hepatocytes, leukocytes including mononuclear leukocytes, stem cellssuch as haemopoetic, neural, skin, lung, kidney, liver and myocyte stemcells (for use in screening for differentiation and de-differentiationfactors), osteoclasts, chondrocytes and other connective tissue cells,keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes.Suitable cells also include known research cells, including, but notlimited to, Jurkat T cells, NIH3T3 cells, CHO, COS, etc. See the ATCCcell line catalog, hereby expressly incorporated by reference.

In one embodiment, the cells may be genetically engineered, that is,contain exogeneous nucleic acid

There are a number of widely available assays to detect either theclustering or activation in a cell. For example, to detect clustering,receptor element ligands and/or receptor-specific antibodies can belabeled, and these labels detected to visualize clustering of receptorelements. In one example of this type of assay, a cell comprising thereceptor element of interest is contacted with a receptor-specific mouseantibody. The receptor-specific mouse antibody is then labeled by theaddition of TRITC-conjugated donkey anti-mouse antibody. Using confocalscanning laser microscopy, fluorescence emitted from the TRITC can bedetected to identify the location of the receptor elements. (VanSteensel, et al., J Cell Sci 108, 3003-3011, 1995).

In another technique, the receptor of interest is labeled directly andthe label is detected as an indication of clustering. For example, thecoding region of a particular receptor element can be fused to thecoding sequence of Green Fluorescent Protein (GFP), and the resultantfusion protein is then expressed in a cell. Using confocal scanninglaser microscopy, fluorescence emitted by the GFP can be detected toidentify the location of the receptor elements. (Gensler, et al., Eur.J. Biochem. 268, 2209-2217, 2001)

A receptor element may also be labeled with one member of a label pair,wherein clustering of that receptor with a receptor labeled with theother member of the label pair is required for detection of the labelpair. For example, in bioluminescence resonance transfer, the codingsequence of the receptor element may be fused with either donor Renillaluciferase or acceptor enhanced yellow fluorescence protein. Insituations where homodimer or homo-oligomerization is suspected, two ormore coding sequences of the same receptor will be used, one fused todonor Renilla luciferase, and one fused to acceptor enhanced yellowfluorescence protein. If necessary, further coding sequences can befused to other known acceptor proteins, such as GFP. In situations whereheterodimerization or hetero-oligomerizaion is suspected, the codingsequence of the first receptor element is fused to donor Renillaluciferase and the coding sequence of the second receptor element isfused to acceptor enhanced yellow fluorescence protein. Again, Ifnecessary, further coding sequences can be fused to other known acceptorproteins, such as GFP. Using confocal laser microscopy, fluorescenceemitted by the acceptor protein can be detected to identify clusteringof the receptor elements. (Kroeger, et al., J Biol Chem 276:16,12736-12743, Apr. 20, 2001).

As will be appreciated by one of skill in the art, the ability of areceptor to cluster can be determined using any of the above techniques,in addition to other techniques known in the art.

The methods and compositions of the instant invention provide BEs whichcomprise a label or tag. By label is meant a molecule that can bedirectly (i.e., a primary label) or indirectly (i.e., a secondary label)detected; for example a label can be visualized and/or measured orotherwise identified so that its presence or absence can be known. Acompound can be directly or indirectly conjugated to a label whichprovides a detectable signal, e.g. radioisotope, fluorescers, enzyme,antibodies, particles such as magnetic particles, chemiluminescers, orspecific binding molecules, etc. Specific binding molecules includepairs, such as biotin and streptavidin, digoxin and antidigoxin etc.Preferred labels include, but are not limited to, fluorescent labels,label enzymes and radioisotopes.

In general, labels fall into four classes: a) isotopic labels, which maybe radioactive or heavy isotopes; b) magnetic, electrical, thermallabels; c) colored or luminescent dyes or moieties; and d) bindingpartners. Labels can also include enzymes (horseradish peroxidase, etc.)and magnetic particles. In a preferred embodiment, the detection labelis a primary label. A primary label is one that can be directlydetected, such as a fluorophore.

Preferred labels include chromophores or phosphors but are preferablyfluorescent dyes or moieties. Fluorophores can be either “smallmolecule” fluores, or proteinaceous fluores.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 andOregon green. Suitable optical dyes are described in the 1996 MolecularProbes Handbook by Richard P. Haugland, hereby expressly incorporated byreference. Suitable fluorescent labels also include, but are not limitedto, green fluorescent protein (GFP; Chalfie, et al., Science 263(5148):802-805 (Feb 11, 1994); and EGFP; Clontech—Genbank AccessionNumber U55762), blue fluorescent protein (BFP; 1. QuantumBiotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor,Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H. Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182(1996)), enhanced yellow fluorescent protein (EYFP; 1. ClontechLaboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303),luciferase (Ichiki, et al., J. Immunol. 150 (12):5408-5417 (1993)),β-galactosidase (Nolan, et al., Proc Natl Acad Sci USA 85 (8):2603-2607(Apr 1988)) and Renilla WO 92/15673; WO 95/07463; WO 98/14605; WO98/26277; WO 99/49019; U.S. Pat. No. 5,292,658; U.S. Pat. No. 5,418,155;U.S. Pat. No. 5,683,888; U.S. Pat. No. 5,741,668; U.S. Pat. No.5,777,079; U.S. Pat. No. 5,804,387; U.S. Pat. No. 5,874,304; U.S. Pat.No. 5,876,995; and U.S. Pat. No. 5,925,558). All of the above-citedreferences are expressly incorporated herein by reference.

Particularly preferred labels for use in the present invention include:Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488,Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633,Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow andR-phycoerythrin (PE) (Molecular Probes) (Eugene, Oreg.), FITC,Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7(Amersham Life Science, Pittsburgh, Pa.). Tandem conjugate protocols forCy5PE, Cy5.5PE, Cy7PE, Cy5.5APC, Cy7APC can be found at drmr.com/abconQuantitation of fluorescent probe conjugation may be assessed todetermine degree of labeling and protocols including dye spectralproperties can be found at metazoa.com/UPL3419.

In another preferred embodiment, the fluorescent label is a GFP and,more preferably, a renilla, ptilosarcus, or aequorea species of GFP.

In a preferred embodiment, a secondary detectable label is used. Asecondary label is one that is indirectly detected; for example, asecondary label can bind or react with a primary label for detection,can act on an additional product to generate a primary label (e.g.enzymes), etc. Secondary labels include, but are not limited to, one ofa binding partner pair; chemically modifiable moieties; nucleaseinhibitors, enzymes such as horseradish peroxidase, alkalinephosphatases, lucifierases, etc.

In a preferred embodiment, the secondary label is a binding partnerpair. For example, the label may be a hapten or antigen, which will bindits binding partner. For example, suitable binding partner pairsinclude, but are not limited to: antigens (such as proteins (includingpeptides) and small molecules) and antibodies (including fragmentsthereof (FAbs, etc.)); proteins and small molecules, includingbiotin/streptavidin; enzymes and substrates or inhibitors; otherprotein-protein interacting pairs; receptor-ligands; and carbohydratesand their binding partners. Nucleic acid-nucleic acid binding proteinspairs are also useful. Preferred binding partner pairs include, but arenot limited to, biotin (or imino-biotin) and streptavidin, digeoxininand Abs, and Prolinx™ reagents (see www.prolinxinc.com/ie4/home.hmtl).

In a preferred embodiment, the binding partner pair comprises an antigenand an antibody that will specifically bind to the antigen. By“specifically bind” herein is meant that the partners bind withspecificity sufficient to differentiate between the pair and othercomponents or contaminants of the system. The binding should besufficient to remain bound under the conditions of the assay, includingwash steps to remove non-specific binding. In some embodiments, thedissociation constants of the pair will be less than about 10-4-10-6M-1, with less than about 10-5 to 10-9 M-1 being preferred and less thanabout 10-7 -10-9 M-1 being particularly preferred.

In a preferred embodiment, the secondary label is a chemicallymodifiable moiety. In this embodiment, labels comprising reactivefunctional groups are incorporated into the molecule to be labeled. Thefunctional group can then be subsequently labeled (e.g. either before orafter the assay) with a primary label. Suitable functional groupsinclude, but are not limited to, amino groups, carboxy groups, maleimidegroups, oxo groups and thiol groups, with amino groups and thiol groupsbeing particularly preferred. For example, primary labels containingamino groups can be attached to secondary labels comprising aminogroups, for example using linkers as are known in the art; for example,homo-or hetero-bifunctional linkers as are well known (see 1994 PierceChemical Company catalog, technical section on cross-linkers, pages155-200, incorporated herein by reference).

In preferred embodiments, multiple fluorescent labels are employed inthe methods and compositions of the present invention. In a preferredembodiment, at least two fluorescent labels are used which are membersof a fluorescence resonance energy transfer (FRET) pair.

FRET is phenomenon known in the art wherein excitation of onefluorescent dye is transferred to another without emission of a photon.A FRET pair consists of a donor fluorophore and an acceptor fluorophore.The fluorescence emission spectrum of the donor and the fluorescenceabsorption spectrum of the acceptor must overlap, and the two moleculesmust be in close proximity. The distance between donor and acceptor atwhich 50% of donors are deactivated (transfer energy to the acceptor) isdefined by the Förster radius (Ro), which is typically 10-100A. Changesin the fluorescence emission spectrum comprising FRET pairs can bedetected, indicating changes in the number of that are in closeproximity (i.e., within 100 Å of each other). This will typically resultfrom the binding or dissociation of two molecules, one of which islabeled with a FRET donor and the other of which is labeled with a FRETacceptor, wherein such binding brings the FRET pair in close proximity.Binding of such molecules will result in an increased fluorescenceemission of the acceptor and/or quenching of the fluorescence emissionof the donor.

FRET pairs (donor/acceptor) useful in the invention include, but are notlimited to, EDANS/fluorescien, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/LC Red 640, fluorescein/Cy5, fluorescein/Cy 5.5 and fluorescein/LC Red 705.

In another aspect of FRET, a fluorescent donor molecule and anonfluorescent acceptor molecule (“quencher”) may be employed. In thisapplication, fluorescent emission of the donor will increase whenquencher is displaced from close proximity to the donor and fluorescentemission will decrease when the quencher is brought into close proximityto the donor. Useful quenchers include, but are not limited to, TAMRA,DABCYL, QSY 7 and QSY 33. Useful fluorescent donor/quencher pairsinclude, but are not limited to EDANS/DABCYL, Texas Red/DABCYL,BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL andfluorescein/QSY 7 dye.

The skilled artisan will appreciate that FRET and fluorescence quenchingallow for monitoring of binding of labeled molecules over time,providing continuous information regarding the time course of bindingreactions.

Preferably, changes in the degree of FRET are determined as a functionof the change in the ratio of the amount of fluorescence from the donorand acceptor moieties, a process referred to as “ratioing.” Changes inthe absolute amount of substrate, excitation intensity, and turbidity orother background absorbances in the sample at the excitation wavelengthaffect the intensities of fluorescence from both the donor and acceptorapproximately in parallel. Therefore the ratio of the two emissionintensities is a more robust and preferred measure of cleavage thaneither intensity alone.

The ratio-metric fluorescent reporter system described herein hassignificant advantages over existing reporters for protein integrationanalysis, as it allows sensitive detection and isolation of bothexpressing and non-expressing single living cells. In a preferredembodiment, the assay system uses a non-toxic, non-polar fluorescentsubstrate which is easily loaded and then trapped intracellularly.Modification of the fluorescent substrate by a cognate protein yields afluorescent emission shift as substrate is converted to product. Becausethe reporter readout is ratiometric it is unique among reporter proteinassays in that it controls for variables such as the amount of substrateloaded into individual cells. The stable, easily detected, intracellularreadout eliminates the need for establishing clonal cell lines prior toexpression analysis. This system and other analogous flow sortingsystems can be used to isolate cells having a particular receptorelement clustering and/or activation profile from pools of millions ofviable cells.

The methods and composition of the present invention may also make useof label enzymes. By label enzyme is meant an enzyme which may bereacted in the presence of a label enzyme substrate which produces adetectable product. Suitable label enzymes for use in the presentinvention include but are not limited to, horseradish peroxidase,alkaline phosphatase and glucose oxidase. Methods for the use of suchsubstrates are well known in the art. The presence of the label enzymeis generally revealed through the enzyme's catalysis of a reaction witha label enzyme substrate, producing an identifiable product. Suchproducts may be opaque, such as the reaction of horseradish peroxidasewith tetramethyl benzedine, and may have a variety of colors. Otherlabel enzyme substrates, such as Luminol (available from Pierce ChemicalCo.), have been developed that produce fluorescent reaction products.Methods for identifying label enzymes with label enzyme substrates arewell known in the art and many commercial kits are available. Examplesand methods for the use of various label enzymes are described in Savageet al., Previews 247:6-9 (1998), Young, J. Virol. Methods 24:227-236(1989), which are each hereby incorporated by reference in theirentirety.

By radioisotope is meant any radioactive molecule. Suitableradioisotopes for use in the invention include, but are not limited to¹⁴C, ³H, ³²P, ³³P, ³⁵S, ¹²⁵I, and ¹³¹I. The use of radioisotopes aslabels is well known in the art.

As mentioned, labels may be indirectly detected, that is, the tag is apartner of a binding pair. By “partner of a binding pair” is meant oneof a first and a second moiety, wherein the first and the second moietyhave a specific binding affinity for each other. Suitable binding pairsfor use in the invention include, but are not limited to,antigens/antibodies (for example, digoxigenin/anti-digoxigenin,dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl,Fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, andrhodamine anti-rhodamine), biotin/avid (or biotin/streptavidin) andcalmodulin binding protein (CBP)/calmodulin. Other suitable bindingpairs include polypeptides such as the FLAG-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner etal., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)] and the antibodies each thereto. As will beappreciated by those in the art, binding pair partners may be used inapplications other than for labeling, as is described herein.

As will be appreciated by those in the art, a partner of one bindingpair may also be a partner of another binding pair. For example, anantigen (first moiety) may bind to a first antibody (second moiety)which may, in turn, be an antigen for a second antibody (third moiety).It will be further appreciated that such a circumstance allows indirectbinding of a first moiety and a third moiety via an intermediary secondmoiety that is a binding pair partner to each.

As will be appreciated by those in the art, a partner of a binding pairmay comprise a label, as described above. It will further be appreciatedthat this allows for a tag to be indirectly labeled upon the binding ofa binding partner comprising a label. Attaching a label to a tag whichis a partner of a binding pair, as just described, is referred to hereinas “indirect labeling”.

By “surface substrate binding molecule” or “attachment tag” andgrammatical equivalents thereof is meant a molecule have bindingaffinity for a specific surface substrate, which substrate is generallya member of a binding pair applied, incorporated or otherwise attachedto a surface.

Suitable surface substrate binding molecules and their surfacesubstrates include, but are not limited to poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags and Nickel substrate; theGlutathione-S Transferase tag and its antibody substrate (available fromPierce Chemical); the flu HA tag polypeptide and its antibody 12CA5substrate [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; thec-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibody substratesthereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616(1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody substrate [Paborsky et al., Protein Engineering, 3 (6):547-553(1990)]. In general, surface binding substrate molecules useful in thepresent invention include, but are not limited to, polyhistidinestructures (His-tags) that bind nickel substrates, antigens that bind tosurface substrates comprising antibody, haptens that bind to avidinsubstrate (e.g., biotin) and CBP that binds to surface substratecomprising calmodulin.

Production of antibody-embedded substrates is well known; see Slinkin etal., Bioconj. Chem. 2:342-348 (1991); Torchilin et al., supra;Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992); King et al., CancerRes. 54:6176-6185 (1994); and Wilbur et al., Bioconjugate Chem.5:220-235 (1994) (all of which are hereby expressly incorporated byreference), and attachment of or production of proteins with antigens isdescribed above. Calmodulin-embedded substrates are commerciallyavailable, and production of proteins with CBP is described in Simcox etal., Strategies 8:40-43 (1995), which is hereby incorporated byreference in its entirety.

As will be appreciated by those in the art, tag-components of theinvention can be made in various ways, depending largely upon the formof the tag. Components of the invention and tags are preferably attachedby a covalent bond.

The production of tag-polypeptides by recombinant means when the tag isalso a polypeptide is described below. Production of tag-labeledproteins is well known in the art and kits for such production arecommercially available (for example, from Kodak and Sigma). Examples oftag labeled proteins include, but are not limited to, a Flag-polypeptideand His-polypeptide. Methods for the production and use of tag-labeledproteins are found, for example, in Winston et al., Genes and Devel.13:270-283 (1999), incorporated herein in its entirety, as well asproduct handbooks provided with the above-mentioned kits.

Biotinylation of target molecules and substrates is well known, forexample, a large number of biotinylation agents are known, includingamine-reactive and thiol-reactive agents, for the biotinylation ofproteins, nucleic acids, carbohydrates, carboxylic acids; see chapter 4,Molecular Probes Catalog, Haugland, 6th Ed. 1996, hereby incorporated byreference. A biotinylated substrate can be attached to a biotinylatedcomponent via avidin or streptavidin. Similarly, a large number ofhaptenylation reagents are also known (Id.).

Methods for labeling of proteins with radioisotopes are known in theart. For example, such methods are found in Ohta et al., Molec. Cell3:535-541 (1999), which is hereby incorporated by reference in itsentirety.

Production of proteins having tags by recombinant means is well known,and kits for producing such proteins are commercially available. Forexample, such a kit and its use is described in the QIAexpress Handbookfrom Qiagen by Joanne Crowe et al., hereby expressly incorporated byreference.

The functionalization of labels with chemically reactive groups such asthiols, amines, carboxyls, etc. is generally known in the art. In apreferred embodiment, the tag is functionalized to facilitate covalentattachment. The covalent attachment of the tag may be either direct orvia a linker. In one embodiment, the linker is a relatively shortcoupling moiety, that is used.to attach the molecules. A coupling moietymay be synthesized directly onto a component of the invention andcontains at least one functional group to facilitate attachment of thetag. Alternatively, the coupling moiety may have at least two functionalgroups, which are used to attach a functionalized component to afunctionalized tag, for example. In an additional embodiment, the linkeris a polymer. In this embodiment, covalent attachment is accomplishedeither directly, or through the use of coupling moieties from thecomponent or tag to the polymer. In a preferred embodiment, the covalentattachment is direct, that is, no linker is used. In this embodiment,the component preferably contains a functional group such as acarboxylic acid which is used for direct attachment to thefunctionalized tag. It should be understood that the component and tagmay be attached in a variety of ways, including those listed above. In apreferred embodiment, the tag is attached to the amino or carboxlterminus of the polypeptide. As will be appreciated by those in the art,the above description of the covalent attachment of a label applies tothe attachment of virtually any two molecules of the present disclosure.

In a preferred embodiment, the tag is functionalized to facilitatecovalent attachment, as is generally outlined above. Thus, a widevariety of tags are commercially available which contain functionalgroups, including, but not limited to, isothiocyanate groups, aminogroups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonylhalides, all of which may be used to covalently attach the tag to asecond molecule, as is described herein. The choice of the functionalgroup of the tag will depend on the site of attachment to either alinker, as outlined above or a component of the invention. Thus, forexample, for direct linkage to a carboxylic acid group of a protein,amino modified or hydrazine modified tags will be used for coupling viacarbodiimide chemistry, for example using1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as is known in theart (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; seealso the Pierce 1994 Catalog and Handbook, pages T-155 to T-200, both ofwhich are hereby incorporated by reference). In one embodiment, thecarbodiimide is first attached to the tag, such as is commerciallyavailable for many of the tags described herein.

Antibody conjugation may be performed using standard procedures(drmr.com.abcon) or by using protein-protein/protein-dye crosslinkingkits from Molecular Probes (Eugene, Oreg.).

In an additional embodiment, the invention provides an activationstate-specific antibody. The methods and compositions of the presentinvention may be used to detect any particular receptor element isoformin a sample that is antigenically detectable and antigenicallydistinguishable from other isoforms of the receptor element which arepresent in the sample. For example, as demonstrated (see, e.g., theExamples) and described herein, the activation state-specific antibodiesof the present invention can be used in the present methods to identifydistinct signaling cascades of a subset or subpopulation of complex cellpopulations; and the ordering of protein activation (e.g., kinaseactivation) in potential signaling hierarchies. Further, in the methodsof the present invention, the use of flow cytometry, particularlypolychromatic flow cytometry, permits the multi-dimensional analysis andfunctional assessment of the signaling pathway in single cells.

As used herein, the term “activation state-specific antibody” or“activation state antibody” or grammatical equivalents thereof, refer toan antibody that specifically binds to a corresponding and specificantigen. Preferably, the corresponding and specific antigen is aspecific isoform of an activable receptor element. Also preferably, thebinding of the activation state-specific antibody is indicative of aspecific activation state of a specific activatable receptor element.

Thus, in preferred embodiments, the binding of an activationstate-specific antibody to a corresponding isoform of an activablereceptor element is indicative of the identity of the activatablereceptor element and of the activation state of the activatable receptorelement.

In a preferred embodiment, the activation state-specific antibody is apeptide comprising a recognition structure that binds to a targetstructure on an activatable receptor element. A variety of recognitionstructures are well known in the art and can be made using methods knownin the art, including by phage display libraries (see e.g., Gururaja etal. Chem. Biol. (2000) 7:515-27; Houimel et al., Eur. J. Immunol. (2001)31:3535-45; Cochran et al. J. Am. Chem. Soc. (2001) 123:625-32; Houimelet al. Int. J. Cancer (2001) 92:748-55, each incorporated herein byreference). In a preferred embodiment, the activation state-specificantibody comprises the following recognition structure: SKVILFE-randompeptide loop-SKVILFE. Antibodies having such recognition structures canbind with high affinity to specific target structures. Further,fluorophores can be attached to such antibodies for use in the methodsof the present invention.

A variety of recognitions structures are known in the art (e.g., Cochranet al., J. Am. Chem. Soc. (2001) 123:625-32; Boer et al., Blood (2002)100:467-73, each expressly incorporated herein by reference)) and can beproduced using methods known in the art (see e.g., Boer et al., Blood(2002) 100:467-73; Gualillo et al., Mol. Cell Endocrinol. (2002)190:83-9, each expressly incorporated herein by reference)), includingfor example combinatorial chemistry methods for producing recognitionstructures such as polymers with affinity for a target structure on anactivable protein (see e.g., Barn at al., J. Comb. Chem. (2001)3:534-41; Ju et al., Biotechnol. (1999) 64:232-9, each expresslyincorporated herein by reference). In another preferred embodiment, theactivation state-specific antibody is a protein that only binds to anisoform of a specific activatable receptor element that isphosphorylated and does not bind to the isoform of this activablereceptor element when it is not phosphorylated or nonphosphorylated. Inanother preferred embodiment the activation state-specific antibody is aprotein that only binds to an isoform of an activatable receptor elementthat is intracellular and not extracellular, or vice versa.

In a preferred embodiment, the recognition structure is an anti-lamininsingle-chain antibody fragment (scFv) (see e.g., Sanz et al., GeneTherapy (2002) 9:1049-53; Tse at al., J. Mol. Biol. (2002) 317:85-94,each expressly incorporated herein by reference).

As used herein, an “activatable receptor element” or “substrate” orgrammatical equivalents thereof, refers to a receptor element that hasat least one isoform (and in some cases two or more isoforms) thatcorresponds to a specific form of the receptor element having aparticular biological, biochemical, or physical property, e.g., anenzymatic activity, a modification (e.g., post-translationalmodification), or a conformation. The activable receptor element can beactivated or nonactivated with respect to a particular biologicalactivity, modification, or conformation. Specifically, the “activated”or “active” form of the activatable receptor eleement has the particularbiological activity, modification, or conformation; whereas the“non-activated” or “non-active” form of the activatable receptor elementdoes not have (or has a lesser or diminished level of) the particularbiological activity, modification, or conformation, respectively. Insome embodiments, there may be more than one isoform associated withactivity or activation state; for example, there may be an isoformassociated with an “open” conformation available for substrate binding,a second “transition state” isoform, and an isoform devoid of activity(e.g., where the activity is inhibited).

In a preferred embodiment, the biological, biochemical, or physicalproperty (e.g. enzymatic activity, modification, or conformation) of theactivatable receptor element is inducible or “activatable” by anactivating agent or by cell signaling events. Examples of activatingagents include, but are not limited to, kinases, phosphatases, proteases(e.g., caspases), and hormones. Examples of cell signaling eventsinclude, but are not limited to, receptor clustering or binding of acognate molecule or ligand.

As used herein, an “isoform” or grammatical equivalents thereof, refersto a form of an activatable receptor having a specific, and preferablydetectable, biological activity, modification, or conformation. Theisoform can be an activated (or active) form, or non-activated (or notactive) form of an activatable receptor. As mentioned, in preferredembodiments, the binding of an activation state-specific antibody to acorresponding isoform of an activable receptor element is indicative ofthe identity of the activatable receptor element and of the activationstate of the activatable receptorelement. In a preferred embodiment, theinvention provides methods for determining a receptor element isoformprofile which comprise determining the presence of an isoform of anactivatable receptor element that is activated (or activated isoform).

In a preferred embodiment, the activated isoform or activated state ofan activable receptor element is a form of the activable receptor havinga particular or specific biological, biochemical, or physical propertythat is not possessed by at least one other isoform of activatablereceptor element. Examples of such properties include, but are notlimited to, enzymatic activity (e.g., kinase activity and proteaseactivity), and receptor element binding activity. Thus, such particularor specific properties or activities are associated with an activatedisoform of an activatable receptor element. Such properties oractivities are sometimes referred to herein as activation stateactivities.

An example of activation state activity is kinase activity for anactivated receptor element. As used herein, a receptor element withprotein kinase activity may refer to a receptor element that whenactivated is capable of catalyzing the phosphorylation of amino acids,or derivatives thereof, which possess an hydroxyl group. Preferredkinases are those which are capable of catalyzing the phosphorylation ofserine, threonine, and tyrosine residues. Kinase activity may bedetermined by supplying a substrate for phosphorylation by kinase, asource of phosphate usable by kinase, and determining thephosphorylation of substrate in the presence of kinase.

Another example of activation state activity is protease activity for anactivated receptor element. As used herein, a receptor element withprotease activity may refer to a receptor element that when activated iscapable of hydrolyzing a peptide bond within a polypeptide comprising anamino acid sequence.

The antigenicity of an activated isoform of an activatable receptorelement is distinguishable from the antigenicity of non-activatedisoform of an activatable receptor element or from the antigenicity ofan isoform of a different activation state. In a preferred embodiment,an activated isoform of a receptor element possesses an epitope that isabsent in a non-activated isoform of a receptor element, or vice versa.In another preferred embodiment, this difference is due to covalentaddition of moieties to a receptor element, such as phosphate moieties,or due to a structural change in a receptor element, as through proteincleavage, or due to an otherwise induced conformational change in areceptor element which causes the element to present the same sequencein an antigenically distinguishable way. In another preferredembodiment, such a conformational change causes an activated isoform ofa receptor element to present at least one epitope that is not presentin a non-activated isoform, or to not present at least one epitope thatis presented by a non-activated isoform of the element. In someembodiments, the epitopes for the distinguishing antibodies are centeredaround the active site of the receptor element, although as is known inthe art, conformational changes in one area of a receptor element maycause alterations in different areas of the element as well.

Many antibodies, many of which are commercially available (for example,see Cell Signaling Technology, www.cellsignal.com, the contents whichare incorporated herein by reference). have been produced whichspecifically bind to the phosphorylated isoform of a protein but do notspecifically bind to a non-phosphorylated isoform of a protein. Manysuch antibodies have been produced for the study of signal transducingproteins which are reversibly phosphorylated. Particularly, many suchantibodies have been produced which specifically bind to phosphorylated,activated isoforms of protein kinases and are sometimes referred toherein as kinase activation state antibodies or grammatical equivalentsthereof. Particularly preferred antibodies for use in the presentinvention include: phospho-AKT Ser473 monoclonal anti-4E2,phospho-p44/42 MAP kinase (Thr202/Tyr204) monoclonal antibody,phospho-TYK2 (Tyr1054/1055) antibody, phospho-p38 MAP kinase(Thr180/Tyr182) monoclonal antibody 28B10, phospho-PKC-PAN substrateantibody, phospho-PKA-substrate, phospho-SAPK/JNK (Thr183/Tyr185) G9monoclonal antibody, phospho-tyrosine monoclonal antibody (P-tyr-100),p44/42 MAPK, p38 MAPK, JNK/SAPK, and phospho-AKT-Thr308.

The present invention provides methods for the determination of areceptor element with kinase activity activation state profile for asample which comprise simultaneously determining the presence ofactivated isoforms of a multiplicity of receptor elements using amultiplicity of antibodies that specifically bind to active,phosphorylated isoforms of the multiplicity of receptor elements.

Additional means for determining kinase activation are provided by thepresent invention. Substrates that are specifically recognized byprotein kinases and phosphorylated thereby are known. Antibodies thatspecifically bind to such phosphorylated substrates but do not bind tosuch non-phosphorylated substrates (phospho-substrate antibodies) may beused to determine the presence of activated kinase in a sample.

In a further embodiment, a receptor element activation and profile isdetermined using a multiplicity of activation state antibodies that areimmobilized. Antibodies may be non-diffusibly bound to an insolublesupport having isolated sample receiving areas (e.g. a microtiter plate,an array, etc.). The insoluble supports may be made of any compositionto which the compositions can be bound, is readily separated fromsoluble material, and is otherwise compatible with the overall method ofscreening. The surface of such supports may be solid or porous and ofany convenient shape. Examples of suitable insoluble supports includemicrotiter plates, arrays, membranes, and beads. These are typicallymade of glass, plastic (e.g., polystyrene), polysaccharides, nylon ornitrocellulose, teflon™, etc. Microtiter plates and arrays areespecially convenient because a large number of assays can be carriedout simultaneously, using small amounts of reagents and samples. In somecases magnetic beads and the like are included.

The particular manner of binding of the composition is not crucial solong as it is compatible with the reagents and overall methods of theinvention, maintains the activity of the composition and isnondiffusable. Preferred methods of binding include the use ofantibodies (which do not sterically block either the ligand binding siteor activation sequence when the protein is bound to the support), directbinding to “sticky” or ionic supports, chemical crosslinking, thesynthesis of the antibody on the surface, etc. Following binding of theantibody, excess unbound material is removed by washing. The samplereceiving areas may then be blocked through incubation with bovine serumalbumin (BSA), casein or other innocuous protein or other moiety.

In a preferred embodiment, an epitope-recognizing fragment of anactivation state antibody rather than the whole antibody is used. Inanother preferred embodiment, the-epitope-recognizing fragment isimmobilized. In another preferred embodiment, the antibody light chainwhich recognizes an epitope is used. A recombinant nucleic acid encodinga light chain gene product which recognizes an epitope may be used toproduce such an antibody fragment by recombinant means well known in theart.

Using the example of two activation state specific antibodies, by“uniquely labeled” is meant that a first activation state antibodyrecognizing a first activated receptor element comprises a first label,and second activation state antibody recognizing a second activatedreceptor element comprises a second label, wherein the first and secondlabel are detectable and distinguishable, making the first antibody andthe second antibody uniquely labeled.

Non-activation state antibodies may also be used in the presentinvention. In a preferred embodiment, non-activation state antibodiesbind to epitopes in both activated and non-activated forms of a receptorelement. Such antibodies may be used to determine the amount ofnon-activated plus activated receptor in a sample. In another preferredembodiment, non-activation state antibodies bind to epitopes present innon-activated forms of a receptor but absent in activated forms of areceptor. Such antibodies may be used to determine the amount ofnon-activated receptor in a sample. Both types of non-activation stateantibodies may be used to determine if a change in the amount ofactivation state receptor, for example from samples before and aftertreatment with a candidate bioactive agent as described herein, coincidewith changes in the amount of non-activation state receptor. Forexample, such antibodies can be used to determine whether an increase inactivated receptor is due to activation of non-activation statereceptor, or due to increased expression of the receptor, or both.

In another preferred embodiment, antibodies are immobilized using beadsanalogous to those known and used for standardization in flow cytometry.Attachment of a multiplicity of activation state specific antibodies tobeads may be done by methods known in the art and/or described herein.Such conjugated beads may be contacted with sample, preferably cellextract, under conditions which allow for a multiplicity of activatedreceptor elements, if present, to bind to the multiplicity ofimmobilized antibodies. A second multiplicity of antibodies comprisingnon-activation state antibodies which are uniquely labeled may be addedto the immobilized activation state specific antibody-activated receptorcomplex and the beads may be sorted by FACS on the basis of the presenceof each label, wherein the presence of label indicates binding ofcorresponding second antibody and the presence of correspondingactivated receptor.

In a preferred embodiment, the present invention provides methods fordetermining a receptor element's clustering and/or activation stateprofile for a single cell. The methods comprise sorting cells by FACS onthe basis of the activation state of at least two receptor elements.Activation state-specific antibodies are used to sort cells on the basisof receptor element activation state.

When using fluorescent labeled components in the methods andcompositions of the present invention, it will recognized that differenttypes of fluorescent monitoring systems, e.g., FACS systems, can be usedto practice the invention. Preferably, FACS systems are used or systemsdedicated to high throughput screening, e.g 96 well or greatermicrotiter plates. Methods of performing assays on fluorescent materialsare well known in the art and are described in, e.g., Lakowicz, J. R.,Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983);Herman, B., Resonance energy transfer microscopy, in: FluorescenceMicroscopy of Living Cells in Culture, Part B, Methods in Cell Biology,vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press(1989), pp. 219-243; Turro, N.J., Modern Molecular Photochemistry, MenloPark: Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.

Fluorescence in a sample can be measured using a fluorimeter. Ingeneral, excitation radiation, from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscause the excitation radiation to excite the sample. In response,fluorescent proteins in the sample emit radiation which has a wavelengththat is different from the excitation wavelength. Collection optics thencollect the emission from the sample. The device can include atemperature controller to maintain the sample at a specific temperaturewhile it is being scanned. According to one embodiment, a multi-axistranslation stage moves a microtiter plate holding a plurality ofsamples in order to position different wells to be exposed. Themulti-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer. The computeralso can transform the data collected during the assay into anotherformat for presentation.

A chip analogous to a DNA chip can be used in the methods of the presentinvention. Arrayers and methods for spotting nucleic acid to a chip in aprefigured array are known. In addition, protein chips and methods forsynthesis are known. These methods and materials may be adapted for thepurpose of affixing activation state antibodies to a chip in aprefigured array.

In a preferred embodiment, such a chip comprises a multiplicity ofreceptor element activation state antibodies, and is used to determine areceptor element activation state profile for a sample. In a preferredembodiment, such a sample is a cell extract. In such a method, detectionof activated receptor elements is by “sandwich assay” as known in theart. Briefly, a sample, preferably a cell extract, is passed over a thechip under conditions which allow the multiplicity of immobilizedreceptor element activation state antibodies to simultaneously bind to amultiplicity of activated receptor elements if present in the sample.The immobilized antibody-receptor element complex is optionally washedand contacted with a second plurality of antibodies comprisingnon-activation state antibodies that are capable of specifically bindingto activated receptor elements while the elements are specifically boundto receptor element activation state specific antibodies. Suchnon-activation state specific antibodies specifically bind to activatedreceptor elements via an epitope that is not recognized by the receptorelement activation state specific antibody. Binding of thenon-activation state specific antibodies to the activation stateantibody-activated receptor element complex is determined and revealsthe presence of activated receptor element in sample. As will beappreciated, the determination of binding of second antibody in thesandwich assay can be accomplished in many different ways. Preferably,the multiplicity of non-activation state specific antibodies areuniquely labeled to facilitate detection.

In an alternative embodiment, a chip comprises a multiplicity ofnon-activation state specific antibodies. Such a chip is contacted withsample, preferably cell extract, and a second multiplicity of antibodiescomprising receptor element activation state specific antibodies is usedin the sandwich assay to simultaneously determine the presence of amultiplicity of activated receptor elements in sample. Preferably, themultiplicity of activation state specific antibodies are uniquelylabeled to facilitate detection.

In a preferred embodiment, flow cytometry is used to detectfluorescence. Other methods of detecting fluorescence may also be used,e.g., Quantum dot methods (see, e.g., Goldman et al., J. Am. Chem. Soc.(2002) 124:6378-82; Pathak et al. J. Am. Chem. Soc. (2001) 123:4103-4;and Remacle et al., Proc. Natl. Sci. USA (2000) 18:553-8, each expresslyincorporated herein by reference).

The detecting, sorting, or isolating step of the methods of the presentinvention can entail fluorescence-activated cell sorting (FAGS)techniques, where FACS is used to select cells from the populationcontaining a particular surface marker, or the selection step can entailthe use of magnetically responsive particles as retrievable supports fortarget cell capture and/or background removal. A variety FACS systemsare known in the art and can be used in the methods of the invention(see e.g., WO99/54494, filed Apr. 16, 1999; U.S.A.N. 20010006787, filedJul. 5, 2001, each expressly incorporated herein by reference).

In a preferred embodiment, a FAGS cell sorter (e.g. a FACSVantage™. CellSorter, Becton Dickinson Immunocytometry Systems, San Jose, Calif.) isused to sort and collect cells based on their receptor elementclustering and/or activation profile (positive cells). The cells arefirst contacted with fluorescent-labeled activation state-specificantibodies directed against specific isoforms of specific activatablereceptor elements. In one embodiment, the amount of bound antibody oneach cell can be measured by passing droplets containing the cellsthrough the cell sorter. By imparting an electromagnetic charge todroplets containing the positive cells, the cells can be separated fromother cells. The positively selected cells can then be harvested insterile collection vessels. These cell sorting procedures are describedin detail, for example, in the FACSVantage™. Training Manual, withparticular reference to sections 3-11 to 3-28 and 10-1 to 10-17.

In another embodiment, positive cells can be sorted using magneticseparation of cells based on the presence of an isoform of anactivatable receptor element. In such separation techniques, cells to bepositively selected are first contacted with specific binding element(e.g., an antibody or reagent that binds an isoform of an activatableprotein). The cells are then contacted with retrievable particles (e.g.,magnetically responsive particles) which are coupled with a reagent thatbinds the specific binding element. The cell-binding element-particlecomplex can then be physically separated from non-positive ornon-labeled cells, for example, using a magnetic field. When usingmagnetically responsive particles, the positive or labeled cells can beretained in a container using a magnetic filed while the negative cellsare removed. These and similar separation procedures are described, forexample, in the Baxter Immunotherapy Isolex training manual.

In a preferred embodiment, methods for the determination of a receptorelement activation state profile for a single cell are provided. Themethods comprise providing a population of cells and sorting thepopulation of cells by FACS. Preferably, cells are separated on thebasis of the activation state of at least two receptor elements. In apreferred embodiment, a multiplicity of receptor element activationstate antibodies (sometimes referred to herein as receptor activationstate specific antibodies) are used to simultaneously determine theactivation state of a multiplicity of receptor elements.

In a preferred embodiment, cell sorting by FACS on the basis of theactivation state of at least two receptor elements is combined with adetermination of other FACS readable outputs, such as the presence ofsurface markers, granularity and cell size to provide a correlationbetween the activation state of a multiplicity of receptor elements andother cell qualities measurable by FACS for single cells.

As will be appreciated, the present invention also provides for theordering of receptor element clustering and activation events in signaltransduction. Particularly, the present invention allows the artisan toconstruct a receptor element clustering and activation heirarchy basedon the correlation of levels of clustering and activation of amultiplicity of receptor elements within single cells.

The present invention may also be used to determine the presence ofcellular subsets, based on correlated receptor element activation withincomplex cellular mixtures such as peripheral blood mononuclear cells.These subsets may represent different differentiation or activationstates or different cell lineages or sublineages.

It will also be recognized that a homogeneous cell population isdesirable for studying signal transduction in order that variances insignaling between cells not qualitatively and quantitatively mask signaltransduction events. The ultimate homogeneous system is the single cell.The present invention provides methods for the analysis of signaltransduction in single cells, where the activated state of the signaltransducing receptor element involved is antigenically distinguishablefrom a non-activated state.

As will be appreciated, these methods provide for the identification ofdistinct signaling cascades for both artificial and stimulatoryconditions in complex cell populations, such a peripheral bloodmononuclear cells, or naive and memory lymphocytes.

The methods provided herein may also involve the use of specificinhibitors of particular receptor elements. The methods provided hereinmay also involve the use of other pharmacological inhibitors ofsignaling pathways. These inhibitors may be used as controls to ensurethat antibodies specifically bind to activated isoforms of receptorelements. For example, an inhibitor of a receptor element known tophosphorylate and activate a kinase may be used to inhibitphosphorylation of the kinase and examine whether an antibodyspecifically recognizes a phosphorylated isoform of the kinase.Alternatively, the inhibitors may be used to further probe signalingpathways and correlations in protein activity, particularly in singlecells.

In a preferred embodiment, a method for screening for a bioactive agentcapable of modulating receptor element clustering and activity isprovided which comprises contacting a cell with a candidate bioactiveagent and determining receptor element activation in said cell by cellsorting said cell by FACS.

In a preferred embodiment, the method comprises contacting a pluralityof cells with a plurality of candidate bioactive agents and sorting thecells by FACS on the basis of the clustering and activation of at leastone receptor element.

By “candidate bioactive agent”, “candidate agent”, “candidatemodulator”, “candidate modulating agent”, or “exogeneous compound” orgrammatical equivalents herein is meant any molecule, e.g., protein,small organic molecule, carbohydrates (including polysaccharides),polynucleotide, lipids, etc. Generally a plurality of assay mixtures canbe run in parallel with different agent concentrations to obtain adifferential response to the various concentrations. Typically, one ofthese concentrations can serve as a negative control, i.e., at zeroconcentration or below the level of detection. In addition, positivecontrols can be used.

Candidate agents encompass numerous chemical classes. In a preferredembodiment, the candidate agents are small molecules. In anotherpreferred embodiment, the candidate agents are organic molecules,particularly small organic molecules, comprising functional groupsnecessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more chemical functional groups.

Candidate agents are obtained from a wide variety of sources, as will beappreciated by those in the art, including libraries of synthetic ornatural compounds. As will be appreciated by those in the art, thepresent invention provides a rapid and easy method for screening anylibrary of candidate modulators, including the wide variety of knowncombinatorial chemistry-type libraries.

In a preferred embodiment, candidate agents are synthetic compounds, asdescribed above in connection with binding elements. One advantage ofthe present method is that it is not necessary to characterize thecandidate agent prior to the assay. Using the methods of the presentinvention, any candidate agents can be screened for the ability tomodulate (e.g., increase or decease) the activity of an activatableprotein. In addition, as is known in the art, coding tags using splitsynthesis reactions may be used to essentially identify the chemicalmoieties tested.

Alternatively, a preferred embodiment utilizes libraries of naturalcompounds, as candidate agents, in the form of bacterial, fungal, plantand animal extracts that are available or readily produced.

Additionally, natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means. Known pharmacological agents may be subjected todirected or random chemical modifications, including enzymaticmodifications, to produce structural analogs.

In a preferred embodiment, candidate agents include proteins, nucleicacids, and chemical moieties.

In a preferred embodiment, the candidate agents are proteins, as definedabove. In a preferred embodiment, the candidate agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be tested, asis more fully described below. In this way libraries of prokaryotic andeukaryotic proteins may be made for screening against any number ofcandidate agents. Particularly preferred in this embodiment arelibraries of bacterial, fungal, viral, and mammalian proteins, with thelatter being preferred, and human proteins being especially preferred.

In a preferred embodiment, the candidate agents are peptides of fromabout 2 to about 50 amino acids, with from about 5 to about 30 aminoacids being preferred, and from about 8 to about 20 being particularlypreferred. The peptides may be digests of naturally occurring proteinsas is outlined above, random peptides, or “biased” random peptides. By“randomized” or grammatical equivalents herein is meant that eachnucleic acid and peptide consists of essentially random nucleotides andamino acids, respectively. Since generally these random peptides (ornucleic acids, discussed below) are chemically synthesized, they mayincorporate any nucleotide or amino acid at any position. The syntheticprocess can be designed to generate randomized proteins or nucleicacids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

The library should provide a sufficiently structurally diversepopulation of randomized agents to effect a probabilistically sufficientrange of diversity to allow interaction with a particular activatableprotein. Accordingly, an interaction library must be large enough sothat at least one of its members will have a structure that interactswith an activatable protein or other specific components of the signaltransduction pathway involving the activable protein. Although it isdifficult to gauge the required absolute size of an interaction library,nature provides a hint with the immune response: a diversity of 10⁷-10⁸different antibodies provides at least one combination with sufficientaffinity to interact with most potential antigens faced by an organism.

Published in vitro selection techniques have also shown that a librarysize of 10⁷ to 10⁸ is sufficient to find structures with affinity for atarget. A library of all combinations of a peptide 7 to 20 amino acidsin length, such as generally proposed herein, has the potential to codefor 20⁷ (10⁹) to 20²⁰. Thus, with libraries of 10⁷ to 10⁸ differentmolecules the present methods allow a “working” subset of atheoretically complete interaction library for 7 amino acids, and asubset of shapes for the 20²⁰ library. Thus, in a preferred embodiment,at least 10⁶, preferably at least 10⁷, more preferably at least 10⁸ andmost preferably at least 10⁹ different sequences are simultaneouslyanalyzed in the subject methods. Preferred methods maximize library sizeand diversity.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the bias is towards peptides or nucleic acidsthat interact with known classes of molecules. For example, when thecandidate agent is a peptide, it is known that much of intracellularsignaling is carried out via short regions of polypeptides interactingwith other polypeptides through small peptide domains. For instance, ashort region from the HIV-1 envelope cytoplasmic domain has beenpreviously shown to block the action of cellular calmodulin. Regions ofthe Fas cytoplasmic domain, which shows homology to the mastoparan toxinfrom Wasps, can be limited to a short peptide region with death-inducingapoptotic or G protein inducing functions. Magainin, a natural peptidederived from Xenopus, can have potent anti-tumor and anti-microbialactivity. Short peptide fragments of a protein kinase C isozyme (RPKC),have been shown to block nuclear translocation of βPKC in Xenopusoocytes following stimulation. And, short SH-3 target peptides have beenused as psuedosubstrates for specific binding to SH-3 proteins. This isof course a short list of available peptides with biological activity,as the literature is dense in this area. Thus, there is much precedentfor the potential of small peptides to have activity on intracellularsignaling cascades. In addition, agonists and antagonists of any numberof molecules may be used as the basis of biased randomization ofcandidate modulators as well.

Thus, a number of molecules or protein domains are suitable as startingpoints for the generation of biased randomized candidate modulators. Alarge number of small molecule domains are known, that confer a commonfunction, structure or affinity. In addition, as is appreciated in theart, areas of weak amino acid homology may have strong structuralhomology. A number of these molecules, domains, and/or correspondingconsensus sequences, are known, including, but are not limited to, SH-2domains, SH-3 domains, Pleckstrin, death domains, proteasecleavage/recognition sites, enzyme inhibitors, enzyme substrates, andTraf.

In a preferred embodiment, the candidate modulating agent is apolypeptide. In another preferred embodiment, the polypeptide is acyclic peptide having at least 4 to 20 amino acids. Also in anotherpreferred embodiment, the polypeptide is a catalytically inactivepolypeptide. Examples of catalytically inactive polypeptides include,but are not limited to, catalytically inactive activable proteins and,more specifically a catalytically inactive kinases (e.g., PI3K) orcaspases. In a further aspect, the candidate modulating agent is peptidefragment of an activatable protein, wherein the peptide fragmentcomprises an amino acid sequence that is a subsequence of thefull-length amino acid sequence of the activable protein.

In a preferred embodiment, the candidate agents are nucleic acids asdescribed above in connection with binding elements.

In a preferred embodiment, the candidate agents are organic moieties asdescribed above in connection with binding elements. Chemical (includingenzymatic) reactions may be done on the moieties to form new substratesor candidate agents which can then be tested using the presentinvention.

As will be appreciated by those in the art, it is possible to screenmore than one type of candidate agent at a time, e.g., by combining thecandidate agents in the methods of the present invention. Thus, thelibrary of candidate agents used may include only one type of agent(i.e. peptides), or multiple types (peptides and organic agents).

By “combining” is meant the combining of the various components in areaction mixture in vitro or in a cell in vivo under conditions whichpromote an activity that is detectable using known methods or using themethods of the present invention (e.g., the binding of an antibody to acorresponding antigen or isoform of an activatable protein, oractivation state of an activatable protein).

It is understood by the skilled artisan that the steps of the assaysprovided herein can vary in order. It is also understood, however, thatwhile various options (of compounds, properties selected or order ofsteps) are provided herein, the options are also each providedindividually, and can each be individually segregated from the otheroptions provided herein. Moreover, steps which are obvious and known inthe art that will increase the sensitivity of the assay are intended tobe within the scope of this invention. For example, there may beadditionally washing steps, blocking steps, etc.

In a preferred embodiment, the reaction mixture or cells are containedin a well of a 96 well plate or other commercially available multiwellplate. In an alternate preferred embodiment, the reaction mixture orcells are in a FACS machine. Other multiwell plates useful in thepresent invention include, but are not limited to 384 well plates and1536 well plates. Still other vessels for containing the reactionmixture or cells and useful in the present invention will be apparent tothe skilled artisan.

The addition of the components of the assay for detecting the activationstate or activity of an activatable protein, or modulation of suchactivation state or activity, may be sequential or in a predeterminedorder or grouping under conditions appropriate for the activity that isassayed for. Such conditions are described here and known in the art.Moreover, further guidance is provided below (see, e.g., in theExamples).

In a preferred embodiment, the methods of the invention include the useof liquid handling components.The liquid handling systems can includerobotic systems comprising any number of components. In addition, any orall of the steps outlined herein may be automated; thus, for example,the systems may be completely or partially automated.

As will be appreciated by those in the art, there are a wide variety ofcomponents which can be used, including, but not limited to, one or morerobotic arms; plate handlers for the positioning of microplates;automated lid or cap handlers to remove and replace lids for wells onnon-cross contamination plates; tip assemblies for sample distributionwith disposable tips; washable tip assemblies for sample distribution;96 well loading blocks; cooled reagent racks; microtitler plate pipettepositions (optionally cooled); stacking towers for plates and tips; andcomputer systems.

Fully robotic or microfluidic systems include automated liquid-,particle-, cell- and organism-handling including high throughputpipetting to perform all steps of screening applications. This includesliquid, particle, cell, and organism manipulations such as aspiration,dispensing, mixing, diluting, washing, accurate volumetric transfers;retrieving, and discarding of pipet tips; and repetitive pipetting ofidentical volumes for multiple deliveries from a single sampleaspiration. These manipulations are cross-contamination-free liquid,particle, cell, and organism transfers. This instrument performsautomated replication of microplate samples to filters, membranes,and/or daughter plates, high-density transfers, full-plate serialdilutions, and high capacity operation.

In a preferred embodiment, chemically derivatized particles, plates,cartridges, tubes, magnetic particles, or other solid phase matrix withspecificity to the assay components are used. The binding surfaces ofmicroplates, tubes or any solid phase matrices include non-polarsurfaces, highly polar surfaces, modified dextran coating to promotecovalent binding, antibody coating, affinity media to bind fusionproteins or peptides, surface-fixed proteins such as recombinant proteinA or G, nucleotide resins or coatings, and other affinity matrix areuseful in this invention.

In a preferred embodiment, platforms for multi-well plates, multi-tubes,holders, cartridges, minitubes, deep-well plates, microfuge tubes,cryovials, square well plates, filters, chips, optic fibers, beads, andother solid-phase matrices or platform with various volumes areaccommodated on an upgradable modular platform for additional capacity.This modular platform includes a variable speed orbital shaker, andmulti-position work decks for source samples, sample and reagentdilution, assay plates, sample and reagent reservoirs, pipette tips, andan active wash station.

In a preferred embodiment, thermocycler and thermoregulating systems areused for stabilizing the temperature of heat exchangers such ascontrolled blocks or platforms to provide accurate temperature controlof incubating samples from 0° C. to 100° C.

In a preferred embodiment, interchangeable pipet heads (single ormulti-channel) with single or multiple magnetic probes, affinity probes,or pipetters robotically manipulate the liquid, particles, cells, andorganisms. Multi-well or multi-tube magnetic separators or platformsmanipulate liquid, particles, cells, and organisms in single or multiplesample formats.

In some embodiments, the instrumentation will include a detector, whichcan be a wide variety of different detectors, depending on the labelsand assay. In a preferred embodiment, useful detectors include amicroscope(s) with multiple channels of fluorescence; plate readers toprovide fluorescent, ultraviolet and visible spectrophotometricdetection with single and dual wavelength endpoint and kineticscapability, fluroescence resonance energy transfer (FRET), luminescence,quenching, two-photon excitation, and intensity redistribution; CCDcameras to capture and transform data and images into quantifiableformats; and a computer workstation.

In a preferred embodiment, the detecting is by FACS. In another aspect,the detecting is by high pressure liquid chromatography (HPLC), forexample, reverse phase HPLC, and in a further aspect, the detecting isby mass spectromety.

These instruments can fit in a sterile laminar flow or fume hood, or areenclosed, self-contained systems, for cell culture growth andtransformation in multi-well plates or tubes and for hazardousoperations. The living cells may be grown under controlled growthconditions, with controls for temperature, humidity, and gas for timeseries of the live cell assays. Automated transformation of cells andautomated colony pickers may facilitate rapid screening of desiredcells.

Flow cytometry or capillary electrophoresis formats can be used forindividual capture of magnetic and other beads, particles, cells, andorganisms.

The flexible hardware and software allow instrument adaptability formultiple applications. The software program modules allow creation,modification, and running of methods. The system diagnostic modulesallow instrument alignment, correct connections, and motor operations.The customized tools, labware, and liquid, particle, cell and organismtransfer patterns allow different applications to be performed. Thedatabase allows method and parameter storage. Robotic and computerinterfaces allow communication between instruments.

In a preferred embodiment, the robotic apparatus includes a centralprocessing unit which communicates with a memory and a set ofinput/output devices (e.g., keyboard, mouse, monitor, printer, etc.)through a bus. Again, as outlined below, this may be in addition to orin place of the CPU for the multiplexing devices of the invention. Thegeneral interaction between a central processing unit, a memory,input/output devices, and a bus is known in the art. Thus, a variety ofdifferent procedures, depending on the experiments to be run, are storedin the CPU memory.

These robotic fluid handling systems can utilize any number of differentreagents, including buffers, reagents, samples, washes, assay componentssuch as label probes, etc.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are expressly incorporated by reference intheir entirety, including the parent application U.S. Ser. No.10/193,462, filed July 2002, which claims the benefit of the filingdates of U.S. Ser. No. 60/304,434 and U.S. Ser. No. 60/310,141.

EXAMPLES Example 1

In this Example, using the methods and compositions of the presentinvention, the present inventors (also referred to herein as “we”) showthat Leukocyte Function Antigen-1 (LFA-1) is essential in the formationof immune cell synapses and has a role in the pathophysiology of variousautoimmune diseases. In this Example, using the methods and compositionsof the present invention, the present inventors demonstrate that ICAM-2induced an LFA-1 signal transduction pathway that is linked to receptorclustering and activation by both the microtubule and actincytoskeleton. ICAM-2 exhibited a 21.7 pM/cell binding affinity asdetermined by single cell analysis. ICAM-2/LFA-1 engagement inducedactivation of PKC and a reorganization of both the actin and microtubulecytoskeleton. These events resulted in a Syk dependent activation of thep44/42 MAPK pathway upon cytotoxic T cell effector-target cell bindingvia active LFA-1. ICAM-2 mediated human CD56⁺CD8⁺ perforin release andresultant cytotoxicity to target leukemia cells. In comparison to theother ICAMs, ICAM-3 was found to be most similar to ICAM-2's effect anddissimilar to ICAM-1. In IL-2 pre-activated human PBMC,ICAM-2>ICAM-3>>ICAM-1 in mediating perforin release of a CD56⁺CD8^(med)population. All ICAMs contributed to perforin and granzyme-A loss inCD56⁺CD8^(high) populations. These results identify a specificfunctional consequence for ICAM-2/LFA-1 in subset-specific cytotoxic Tcell immunity.

Introduction

Leukocyte Function Antigen-1 (LFA-1) is an α,β heterodimer integrininvolved in leukocyte adhesion (van Kooyk, Y., and Figdor, C. G. (2000)Curr Opin Cell Biol 12, 542-547). At present, it is well understood thatLFA-1 participates in lymphocyte adhesion, with prominent roles in theformation of the immunological synapse (Dustin, M. L., and Shaw, A. S.(1999) Science 283, 649-650), and lymphocyte extravasation andrecirculation (Volkov, Y., et al., (2001). Nat Immunol 2, 508-514).LFA-1 adhesion is governed by the intercellular adhesion molecule(ICAMs)-1, -2, and -3 ligands (van Kooyk and Figdor, 2000). Patientsafflicted with Leukocyte Adhesion Deficiency disorder (LAD), a syndromein which the LFA-1 integrin is mutated or missing, suffer severrecurrent bacterial infections and impaired overall immunity (Bunting,M., et al., (2002) Curr Opin Hematol 9, 30-35). Among these clinicalmanifestations, the LFA-1 knockout mouse has suggested that LEA-1 mayhave a potential role in mediating tumor regression in adoptiveimmunotherapy (Mukai, S., et al., (1999) Cell Immunol 192, 122-132;Nishimura, T., et al., (1999) J Exp Med 190, 617-627). Although thesestudies genetically link a lymphocyte adhesion molecule with impairedimmune function, the molecular details that mediate theseimmunopathologies are less well understood.

Investigations of LFA-1 have primarily focused on the integrin'sadhesive role. It is unclear as to how the physical processes of LEA-1integrin activation and receptor clustering are interconnected andtranslated into cellular signals upon ligand binding. It is lessunderstood how the absence of these events leads to the devastatingeffects of LAD and the impaired immune responses in LFA-1 knockout mice.We therefore sought to decipher the molecular details of a modelinteraction of ICAM-LFA-1 to understand LEA-1 signaling mechanismsinitiated upon cell-to-cell contact. Utilizing multiparameter singlecell analysis to monitor LFA-1 receptor dynamics upon treatment with asoluble ICAM-2, we found that both the actin and the microtubulecytoskeleton couple ICAM-2 adhesion to LFA-1 activation and clustering.The microtubule cytoskeleton constrained the LFA-1 conformational change(activation), an event that preceded LFA-1 clustering as measured bymultiparameter flow cytometry. The induced LFA-1 activation led to theactivation of the p44/42 Mitogen Activated Protein Kinase pathway (MAPK;RAF/MEK/ERK), an event that was dependent on both Pyk2 and Syk kinaseactivities.

The present inventors investigated these molecular details of the ICAM-2mediated LFA-1 activation in the adhesion between cytotoxic T cells anda target leukemia cell, an event that requires cell-to-cell contact.ICAM-2 stimulation of human CD56⁺CD8⁺ T cells could induceperforin/granzyme-A mediated cytotoxicity of leukemia cells. Thisdirected killing was shared by ICAM-3 and to a lesser extent by ICAM-1,two other LFA-1 ligands. These results distinguish a signaling mechanismfor ICAM-2/LFA-1 directed cytotoxic T lymphocyte immunity and suggestpossible mechanisms by which tumor secretion of ICAM-2 and possiblyICAM-3 might allow for evasion of a directed cytotoxic T cell immuneresponse.

Results Recombinant ICAM-2 Promotes LFA-1 Mediated Adhesion

The present inventors chose a model Jurkat T cell line as a system toinitially dissect the LFA-1 signaling mechanism and then verified thefindings in human T cells. A biochemically purified ICAM-2 protein wasproduced to study ICAM-2/LFA-1 interactions in the absence of otherligands (FIG. 1A-H). We purified human ICAM-2 from retrovirallytransduced N IH3T3 cells using immunoaffinity chromatography andsubsequent gel filtration. We compared it to an ICAM-2-FC fusion proteinproduced in NSO murine myeloma cells. These murine based mammalianexpression systems were chosen on the basis that they yielded abioactive form of ICAM-2. Biochemical analysis of ICAM-2FC protein wasconsistent with the expected molecular weight of the fusion protein (76kD, FIG. 1A) and purified human ICAM-2 displayed a molecular weight of72-74 kD (FIG. 1A). This size was similar to the 75 kD ICAM-2 purifiedfrom Jurkat T cells (data not shown).

The present inventors generated a FITC conjugated ICAM-2 (ICAM-2-FITC)to study LFA-1 receptor dynamics by flow cytometry and laser scanningconfocal microscopy (LSCM) (FIG. 31G). We tested for ligand binding ofthe LFA-1 receptor by monitoring the binding kinetics of ICAM2-FITC onsingle cells. Low binding was observed in the first 150 seconds,whereupon there was a progressive increase until 750 seconds, andleveled thereafter (FIG. 1B, middle panel). In contrast, an a-LFA-1antibody displayed an initial spike in the first 50-100 seconds andequilibrated until 800 seconds (FIG. 1B, bottom panel). Pre-activatingLFA-1 by treatment with PMA (McDowall, A., et al., (1998) J Biol Chem273, 27396-27403) showed an immediate binding of ICAM-2 (FIG. 1C, toppanel). The gradual ICAM-2 binding after 150 seconds suggested anenhanced LFA-1 binding for its ICAM-2 ligand after some binding-inducedevent—a property not observed using the a-LFA-1 or upon PMA activatedLFA-1 (FIG. 1B). Binding of ICAM-2-FITC was not observed in trypsinizedcells (data not shown) and was blocked by antibodies to LFA-1 (describedbelow).Therefore, there appeared to be an increase in binding of theICAM-2 ligand as a function of time, suggesting the presence of aninduced binding site on the target cells.

Analysis of the ICAM-2 binding population by flow cytometry showed adependency on both the actin cytoskeleton and temperature. ICAM-2adhesion was enhanced at 37° C. vs. 4° C. (FIG. 1C). Pre-treatment withthe actin depolymerizing agent cytochalisin D revealed two ICAM-2binding populations at both 37° C. and 4° C., contrasting with thebinding phenomena observed for a-LFA-1 (FIG. 1C). Saturation ofICAM-2-FITC was observed at 37° C. more readily than at 4° C. (data notshown, FIG. 1H). Single cell binding affinity measurements for ICAM-2were obtained by computing the percent ICAM-2-FITC bound per cell (FIG.1D). Curve fit analysis indicated a dissociation constant of 0.21±0.07mM/10 ⁴ cells (FIG. 10). This value equates to 21.7 pM/cell,representing the first ligand binding measurements reported for ICAM-2within the physiological context of cell surface LFA-1. Thus,quantitative single cell analysis of ICAM-2 ligand binding suggestsstrong binding at physiological temperatures.

Soluble ICAM-2 induces LFA-1 clustering and cytoskeleton polarization

The present inventors investigated if LFA engagement alteredcytoskeletal structures and observed a reorganization of both the actinand microtubule cytoskeleton upon ICAM-2 stimulus (FIG. 2A). The presentinventors monitored the cytoskeletal architecture by flow cytometry andobserved a simultaneous change in the actin and microtubule organizationupon ICAM-2 binding (FIG. 2B), an effect consistent withdepolymerization. ICAM-2 treatment induced a rapid clustering of LFA-1within one minute, with multiple clustering events at five minutes (FIG.2C, left panel). Using the ICAM-2-FITC ligand to visualize the cellsurface, indicated that the ICAM-2 ligand induced clustering of theLFA-1 receptor (FIG. 2C, left panel). The clustering event showed somecolocalization using a non-blocking b2 integrin antibody (clone CTB104)(FIG. 2C, right panel). Thus, we speculated that ICAM-2 binding to LFA-1induced a signal that resulted in a reorganization of the LFA-1/ICAM-2complex. We therefore decided to investigate this in relation to theobserved changes in the actin/microtubule cytoskeleton.

The present inventors assessed LFA-1 receptor dynamics by multiparameterflow cytometry upon ICAM-2 binding to correlate LFA-1 activation andclustering. We utilized the doublet discriminator module on aFACSCalibur machine to distinguish between distributed and focalizedfluorescence pulses (FFP) upon laser excitation of single cells.Incubation of ICAM-2 at 37° C. vs. 4° C. displayed a decrease in theFFP, an effect that was greatly enhanced upon cytochalisin D treatment(FIG. 2D). ICAM-2-FITC surface binding was monitored by the fluorescenceintensity and normalized against the time-of-flight (TOF) of thefluorescence pulse (FP). We interpreted the value of ICAM-2-FITCintensity per TOF as a quantitative assessment for LFA-1 clustering, asthe TOF is proportional to the laser-excited cellular area. Computingthis value as a function of time for an ICAM-2 stimulus (FIG. 2E) isproportional to the increased clustering events observed by LSCM (seeFIG. 2C).

ICAM-2 Adhesion Induces a Conformational Change in LFA-1 That isRegulated by the Microtubule Cytoskeleton

Although the enhanced ICAM-2 adhesion and induced LFA-1 clustering isreflective of overall increased avidity for the ICAM-2 ligand, it doesnot necessarily reflect an LFA-1 activation state (high affinity state)(McDowall et al., 1998). Upon LFA-1 activation, a conformational changeexposes an epitope that is recognized by the mAb24 antibody (Neeson, P.J., et al., (2000) J Leukoc Biol 67, 847-855). A mAb24-Alexa633conjugate was used to assess the activation state of LFA-1 upon ICAM-2stimulus by flow cytometry. Unstimulated cells did not display mAb24binding, contrasting the induction observed with PMA treatment (FIG.2F). ICAM-2 stimulated cells displayed a bimodal population in activeLFA-1, an effect that was attenuated by cytochalisin D (FIG. 2F).Treatment with microtubule disrupting agents, nocodazole and taxol,resulted in full activation of LFA-1 upon ICAM-2 stimulus (FIG. 2F). Incontrast, disrupting the actin cytoskeleton via cytochalisin Ddiminished the ICAM-2 induced LFA-1 activation, although it enhancedLFA-1 receptor clustering and subsequent ICAM-2 binding (see FIG. 2D).Therefore, the actin and microtubule cytoskeletal network differentiallyimpact LFA-1 activity and avidity.

The present inventors monitored LFA-1 activation and clusteringsimultaneously as a function of ICAM-2 stimulus per time by flowcytometry. Correlating the mean fluorescence of mAb24 antibody with theLFA-1 clustering value revealed LFA-1 activation preceeded LFA-1clustering (FIG. 2G) within 30 seconds there was a significant increasein binding of the mAb24 antibody but only a modest increase inclustering. However, after another 30 seconds up to 30 minutes therelative binding of mAb24 increased somewhat but there was a significantincrease in the clustering value (FIG. 2G). Thus, these results suggestthat the ICAM-2 ligand induced activation of LFA-1 is followed bysubsequent LFA-1 clustering.

The present inventors observed that treating cells with a PKC inhibitor,bisindolymaleimide I (BIM I), inhibited ICAM-2 induced LFA-1 activationas measured by using mAb24 binding (FIG. 3A). ICAM-2 adhesion, asmeasured by the binding of ICAM-2-FITC, was not affected (FIG. 3A). Thissuggested that the ligand induced receptor conformational change wasdependent on intracellular kinases. Interestingly, ICAM-2 induced acalcium influx, a component necessary in PKC activation (data notshown). Thus, these observations suggest that the ICAM-2 ligand inducedexposure of the mAb24 neoepitope triggers a PKC dependent intracellularsignaling event. We decided to investigate the downstream signalingconsequences of ICAM-2 binding to LFA-1.

ICAM-2 Induces p44/42 MAPK Activity Through LFA-1

Flow cytometric based kinase profiling experiments were performed toidentify a signaling pathway downstream of PKC activation upon ICAM-2stimulus. Treatment with ICAM-2 induced both p44/42 MAPK phosphorylationand activation (FIG. 3B-D). An ICAM-2 titration correlated withphosphorylation of p44/42 MAPK as determined by single cell flowcytometric analysis (FIG. 3B, top panel), results congruent with kinaseactivity analysis (see FIG. 3C). Titration of mAbs to a_(L) and b2integrins competed with ICAM-2 binding, and thus diminished the inducedp44/42 MAPK phosphorylation (FIG. 3B, bottom panel). This inhibition wasnot observed after pretreatment with mAbs to b1, b3, a_(M), or a_(X)integrins (FIG. 3C) indicating that the ICAM-2/LFA-1 interaction wasmediating the p44/42 MAPK activation.

Activation of PKC, PYK2, and SYK Are Necessary for the ICAM-2/LFA-1Induction of p44/42 MAPK Activity

The present inventors undertook flow cytometric based p44/42 MAPK kinaseinhibition and activation profiling to identify necessary components forLFA-1 signaling. PKC inhibitor BIM I, cytoskeletal disrupting agentscytochalisin D, taxol, nocodozole, and sequestering of divalent cationsby EDTA diminished the ICAM-2 induced p44/42 MAPK signal (FIG. 3D),suggesting that the ligand-induced events of LFA-1 are mechanicallylinked to signal transduction by the actin-microtubule cytoskeleton. Toidentify upstream kinases that were responsible for signal transmissionfrom LFA-1 to p44/42 MAPK, a series of kinase inhibitors were appliedand tested for their ability to abrogate the ICAM-2 induced p44/42 MAPKactivity (FIG. 2H-I), whereas Herbimycin A and Emodin, inhibitors of srcand p56lck had no effect. Tyrphostin A9 and piceatannol, specificinhibitors of proline-tyrosine kinase 2 (Pyk2) and Spleen-tyrosinekinase (Syk), respectively (Avdi, et al. (2001) J Biol Chem 276,2189-2199.; Fuortes, et al., (1999) J Clin Invest 104, 327-335)abrogated the ICAM-2 induced activation of p44/42 MAPK and its upstreamactivator Raf-1 (FIG. 4A).

The present inventors tested whether Pyk2 and Syk interacted with the b2integrin. Pyk2 and Syk were phosphorylated and co-immunoprecipitatedwith b2 integrin upon ICAM-2 treatment (FIG. 34B), indicating Pyk2 andSyk translocated to the membrane (FIG. 3E). This was coincident withphosphorylation of Pyk2 and Syk upon ICAM-2 stimulus as a function oftime (FIG. 4B-C). Phosphorylation of PKCa/b_(II), and Pyk2 were detectedat one minute, followed by Syk phosphorylation at 5 minutes (FIG. 4C).We confirmed that Pyk2 and Syk activities were dependent on PKCactivation (data not shown FIG. 4D-E). Taken together with the aboveresults, this suggested that the LFA-1 signaling mechanism imparted byICAM-2 is at least initiated by PKC and relayed to the p44/42 MAPKpathway by Pyk2 and Syk.

LFA-1 is Involved in Effector-Target Cell Adhesion and Facilitates HumanCytotoxic T Cell Activation

Since LFA-1 is involved in adhesion between lymphocytes, a process thatoccurs at several immunological synapses, we were interested ininvestigating the molecular events identified for the ICAM-2/LFA-1interaction in a physiological context. It has been suggested that aclustered topographic presentation of ICAM-2, independent of expressionlevels, is an effective target structure by which natural killer cellsinitiated cytotoxicity (Helander, T. S., et al., (1996) Nature 382,265-268). We first applied a FACS based effector-target killing assay toquantitatively monitor target cell lysis of HL60 leukemic cells upontreatment with stimulated human PBMC at various effector : target cellratios. Flow cytometric detection of target cell lysis has been reportedto be more sensitive than the standard chromium release assays (Lecoeur,H., et al., (2001) J Immunol Methods 253, 177-187). We labeled HL60cells with the fluorescent dye CFSE and monitored the cell quantity byflow cytometry in standard effector-target cell based assays. SolubleICAM-2 could initiate target cell lysis in the presence of IL-2 but notin the absence of IL-2 (FIG. 5A). In IL-2 pre-activated cells, ICAM-1and ICAM-3 did not initiate as potent a cytotoxic cell response incontrast to ICAM-2 (FIG. 5B).

Since natural killer cells (NK) comprise a heterogeneous population,namely specific cytotoxic T lymphocytes (CTL, with C8⁺ subsets therein),NK cells (CD16⁺ and subsets therein), and CD4⁺ TH1 cells (Biron, C. A.,and Brossay, L. (2001) Curr Opin Immunol 13, 458-464.), we determined ifICAM-2 was unique to a particular human NK cell subset. We utilized themultidimensional gating capability of flow cytometry to identifydistinct cellular populations that were contributing to the cytolyticactivity observed in human PBMC. We also monitored intracellular levelsof perforin and granzyme-A by flow cytometry, two proteins that mediatetarget cell lysis by NK cells in these populations. We identified 6distinct populations by CD8 and CD56 surface stains in human PBMC (FIG.36 panel I) and gated on these subsets for all subsequent intracellularfunctional assays (FIG. 36A panels II-V). We performed effector-targetcytotoxicity assays in the presence of ICAM-1, ICAM-2, and ICAM-3soluble ligand and HL60 target cells. We did not observe significantchanges in population subset frequencies post stimulation (FIG. 6A,panel I). The CD56⁺CD8^(low) population displayed no significant changesin intracellular perforin or granzyme-A upon stimulation with ICAM-1,-2, or -3 (FIG. 6A, panel II). The CD56⁺CD8⁺ population displayed aslight increase (1.5-2 fold) in the frequency of the perforin negativepopulation for ICAM-2 and ICAM-3 (21.5% ICAM-2>19.8% ICAM-3>13.7%ICAM-1) (FIG. 6A, panel III). The CD56⁺CD8^(high) population displayed aloss in both granzyme-A and perforin for ICAM-1, -2, -3 stimulationscompared to unstimulated with a significant loss in the granzyme-Anegative population for ICAM-2 (58.3%) compared to ICAM-1 (4.12%) orICAM-3 (3.07%) (FIG. 6A, panel IV). The CD56⁻CD8^(high) also displayed aloss of both granzyme-A and perforin by all ICAM stimulations (FIG. 6Apanel V). Since it was not possible to positively identify the subsetswithin the CD56⁻CD8⁻ population, they were omitted from analysis.

Quantifying the intracellular amounts of perforin and granzyme-A in theCD56CD8 subsets relative to unstimulated cells also identifiedsimilarities and differences for the ICAMs as evidenced below. ICAM-2and ICAM-3 mediated loss of granzyme-A and perforin to a greater extentthan ICAM-1 (FIGS. 6B-C). Additionally, in IL-2 pre-activated cells,differences where seen with the ICAM stimulations: ICAM-2>ICAM-3>>ICAM-1displayed a loss of perforin, particularly in the CD56⁺CD8^(med/high)populations (FIG. 6B). ICAM-2 and ICAM-3 also induced perforin loss inthe CD56⁺CD8^(low), however ICAM-2 required preactivation by IL-2 (FIG.6B). There were lower levels of granzyme-A detected for the CD8^(high)subsets (CD56⁺ or CD56⁻) for ICAM-2>ICAM-3>ICAM-1>unstimulated (FIG.6C). In the presence of IL-2 pre-activation, all the ICAMs inducedrelease of granzyme-A in the CD56⁺CD8^(high/med) populations, with aparticular decrease by ICAM-2 (FIG. 6C). No significant changes wereseen in the CD56⁺CD8^(low) population for granzyme-A (FIG. 6C). Thesedifferences were similar at various effector-target cell ratios (50:1,25:1, 12.5:1) (data not shown). Thus, similarities and difference existfor ICAM-1, -2, and -3 stimulation of cytolytic activity in CD56CD8subsets. All three ICAMs mediated perforin release in theCD56⁻CD8^(high) populations. ICAM-2 and ICAM-3 were most similar inmediating perforin/granzyme-A release in the CD56⁺CD8^(high) andCD56⁺CD8^(med) populations.

We focused on the CD56⁺CD8⁺ cells (both the CD8^(med) and CD8^(high)subsets) and tested if inhibition of Syk, p44/42 MAPK or disruption ofthe cytoskeleton detrimentally affected effector-target (E:T) cellconjugation as measured by a flow cytometric conjugate formation assay(Morgan, M. M., et al., (2001) J Immunol 167, 5708-5718). Disruption ofcytoskeletal actin and microtubules enhanced E:T conjugate formation(FIG. 7A) congruent with prior results that disruption by these agentsenhanced LFA-1 activation. Inhibition of Syk by piceatannol inhibitedconjugate formation whereas inhibiting p44/42 MAPK by PD98059 did not(FIG. 7A). These results suggest that Syk activity is necessary forLFA-1 adhesion of effector-target cells and is consistent with a reportindicating that Syk/ZAP-70 are necessary for LFA-1 to LFA-1 activationon the same cell (Soede, R. D., et al., (1999) J Immunol 163,4253-4261). p44/42 MAPK appeared to not be necessary for E:T conjugateformation. Monitoring active LFA-1 and intracellular activation ofp44/42 depicted a time dependent correlation between these two markersin CD56⁺CD8⁺ cells as stimulated by ICAM-2 (FIG. 7B).

Discussion

In this report it was observed that (1) ICAM-2 can induce LFA-1clustering, activation, and cytoskeletal reorganization in the absenceof exogenous activators such as cytokines or TCR signaling; (2) LFA-1transmits a signal to the p44/42 MAPK pathway involving PKC, Pyk2, andSyk upon ligand binding; and (3) LFA-1 receptor dynamics aremechanically coupled to signal transduction by both the actin andmicrotubule cytoskeleton network. The physiological outcome of thesemolecular events triggered perforin and granzyme A mediated CD56⁺ CD8⁺ Tcell cytotoxicity that were mostly shared by ICAM-2 and ICAM-3 but notICAM-1.

b2 integrin signaling mechanisms vary depending on the system of studyand are centered on adhesive roles in cell morphology and motility (Dib,K. (2000) Front Biosci 5, D438-451). b2 integrin signaling has beenshown to involve cytoskeletal reorganization via tyrosinephosphorylation of paxillin, vav, and GTPase activating proteins amongothers (Fuortes, M., et al., (1994) J Cell Biol 127, 1477-1483; Zheng,L., et al., (1996) Proc Natl Aced Sci USA 93, 8431-8436). Studiesfocused on LFA-1 mediated leukocyte adhesion (CD11a/CD18) have shown aregulatory role for PKC in LFA-1 avidity (Bleijs, D. A., et al., (2001)J Biol Chem 276, 10338-10346; Hedman, H., and Lundgren, E. (1992) JImmunol 149, 2295-2299) and have demonstrated that TCR signaling canactivate LFA-1 (Peterson, E. J., et al., (2001) Science 293, 2263-2265).It has also been shown that chemokines, in the absence of TCR signaling,can serve as activators of LFA-1 during lymphocyte/endothelial contact(Constantin, G., et al., (2000) Immunity 13, 759-769). It has not beenclear how LFA-1 integrin adhesion, clustering, and activation arecoupled to intracellular signaling events, in the absence of external(chemokine) or internal (TCR or costimulatory molecule) stimulation.

A synthesized peptide of ICAM-2's first Ig domain (P1, amino acids21-42) can induce LFA-1 mediated adhesion at high concentrations (62mM), which was comparable to a 48-fold lower ICAM-2 soluble proteinconcentration (1.3 mM) in a bulk cellular adhesion assay (Kotovuori, A.,et al., (1999) J Immunol 162, 6613-6620). However, P1 binding did notinduce the active conformation of LFA-1 and did not induce calciuminflux (Kotovuori et al., 1999), whereas full length ICAM-2 bindingresulted in active LFA-1 (see FIG. 34D) and a calcium influx event (datanot shown). The calculated ICAM-2 affinity of 217±66 nM (per 10⁴ cells)contrasts the 605±55 nM k_(D) reported using BIAcore analysis of anengineered “active” locked I domain of LFA-1 (Shimaoka, M., et al.,(2001) Proc Natl Acad Sci USA 98, 6009-6014). The reported affinitiesfor ICAM-2 binding here take advantage of single cell resolution withina physiological context, something not possible utilizing purified orgenetically engineered LFA-1. The differences observed for peptide vs.protein concentrations are likely attributed to impurities in thepeptide synthesis and/or presence of carbohydrate moieties native to theendogenous ICAM-2, which comprise greater than 30 kD of its approximate66 kD molecular weight and have been suggested to orient ICAM-2 bindingto LFA-1 (Casasnovas, J. M., et al. (1997) Nature 387, 312-315; deFougerolles, A. R., et al. (1991) J Exp Med 174, 253-267).

We investigated the role of the actin and microtubule cytoskeleton inLFA-1 receptor activation and clustering as induced by the ICAM-2 ligandby multiparameter flow cytometry. Disruption of the actin cytoskeletonenhanced LFA-1 clustering and ICAM-2 binding, corroborating previousstudies that suggested the actin cytoskeleton constrains LFA-1 mobility(Lub, M., et al., (1997) Mol Biol Cell 8, 341-351). Interestingly, actindepolymerization abrogated the ICAM-2 induced LFA-1 activation. Incontrast, disruption of the microtubules by both nocodazole and taxolenhanced LFA-1 activation as determined by exposure of the neo-epitoperecognized by the mAb24. Recently, it has been reported thatdepolymerization of microtubules increases the lateral mobility of b2integrins in macrophage cell lines (Zhou, X., et al., (2001) J Biol Chem276, 44762-44769); therefore its conceivable that the microtubulesregulate the conformational change upon ligand binding necessary forexposure of the LFA-1 activation epitope. These observations suggest theactin-microtubule cytoskeleton regulates both the high-avidity and highaffinity state of LFA-1 upon ligand binding. We observed that LFA-1signal transduction was abrogated in the presence of all cytoskeletaldisrupting agents tested (cytochalisin D, nocodazole, and taxol)indicating that the LFA-1 receptor is linked to signal transductionmachinery by the cytoskeleton. Thus, the mechanistic uncoupling of thehigh avidity and high affinity states of LFA-1 suggests thatintracellular events that regulate/mediate these two states exist at theLFA-1 integrin-cytoskeletal juncture and relay the LFA-1 receptordynamics to intracellular signaling proteins upon ligand binding.

Several chemical inhibition screens were designed to identify theproteins involved in the LFA-1 to p44/42 MAPK signaling event. Both Pyk2and Syk were identified to be necessary for activation of the p44/42MAPK pathway and were dependent on PKC activity upon ICAM-2 binding.Phosphorylation of Pyk2 has been associated with homotypic adhesionmediated by an LFA-1/ICAM-1 interaction in B cells (McDonald, J. T., etal., (2000) Immunol Invest 29, 71-80). In addition, Pyk2 activation hasbeen shown to be necessary for p44/42 MAPK activity in other modelsystems (Barsacchi, R., et al., J. (1999) FEBS Lett 461, 273-276.; Lev,S., et al., (1995) Nature 376, 737-745). Syk is a tyrosine kinaseessential in aIIIb3 signaling (Saci, A., et al., (2000) Biochem J 351 Pt3, 669-676), and links FceRI signaling to the ras/MAPK pathway(Jabril-Cuenod, et al., (1996) J Biol Chem 271, 16268-16272). Inhibitionor ablation of Syk, either by pharmacological means (via inhibition bypiceatannol), biochemical means (dominant negative Syk), or geneticmeans (Syk^(−/−) mice) inhibits natural cytotoxicity (Brumbaugh, K. M.,et al., (1997) J Exp Med 186, 1965-1974; Colucci, F., et al., (1999) JImmunol 163, 1769-1774). Thus LFA-1 activation signaling tb Syk, akinase that has been shown to be important for NK cell function,provides a biochemical link between surface integrin activation andeffector cell function.

The present inventors demonstrated that both Pyk2 and Syk are necessaryin ICAM-2 induced LFA-1 signaling to Raf-1, the upstream kinase in thep44/42 MAPK (RAF/MEK/ERK) cascade. Inhibition of p44/42 MAPK did notprevent the occurrence of CD56⁺CD8⁺ cell conjugation. Byimmunofluorescence analysis, it has been shown that treatment of the NKleukemic cell line YT with the p44/42 MAPK inhibitor PD98059 inhibitsperforin redistribution to the site of effector-target cell contact(Wei, S., et al., (1998) J Exp Med 187, 1753-1765). In addition, thep44/42 MAPK pathway has been shown to be important in the regulation ofcytoxicity in natural killer cells (Jiang, K., et al., (2000) NatImmunol 1, 419-425). Thus, the p44/42 MAPK pathway, here demonstrated tobecome active upon LFA-1/ICAM-2 binding, has been shown to be connectedto at least perforin granule exocytosis. Thus, the LFA-1 signalingpathway as elicited by ICAM-2 contains signaling junctures that map toboth the effector-target cell adhesion event and activation of cytolyticmachinery in the human CD56⁺CD8⁺ cytotoxic T cell population. Theseresults provide direct evidence for a functional consequence of LEA-1integrin adhesion with cytolytic signaling mechanisms.

We also observed that ICAM-2 was similar to ICAM-3 in mediatingcytolytic activity as evidenced by release of perforin and granzyme-A ineffector-cell conjugation, effects of which contrasted ICAM-1 (see FIG.38). We have previously observed similarities between ICAM-2 and ICAM-3intracellular signaling mechanism that also differed from that of ICAM-1(Perez, O. D., et al., (2002) Immunity 16, 51-65). However, the resultsdo not exclude the possibility of ICAM-2 stimulating other yet to beidentified cytotoxic capable subsets, as high cytolytic activity wasobserved in bulk PBMC (see FIG. 37).

Prior investigations into cytotoxic T cells have established thatblocking the LFA-1/ICAM interactions inhibits effector-target celladhesion and therefore concluded that it also blocks cytolytic activityin NK cells (Donskov, F., et al., (1996) Nat Immun 15, 134-146; Krensky,A. M., et al., (1984) J Immunol 132, 2180-2182; Matsumoto, G., et al.,(2000) Eur J Immunol 30, 3723-3731). Functional studies of NK cells fromLFA-1^(−/−) mice have demonstrated that LFA-1 adhesion is necessary forIL-2 activated NK killing (Matsumoto et al., 2000) and also thatLFA-1^(−/−) CD8⁺ T cells are defective for T cell activation andeffector function (Shier, P., et al., (1999) J Immunol 163, 4826-4832).Interestingly, NK cell cytotoxicity is defective in NK cells from LADpatients (Shibuya, K., et al., (1999) Immunity 11, 615-623). It has onlyrecently been shown that the directed killing of cytotoxic T lymphocytesinvolves polarization of the microtubule-organizing center (MTOC)towards LFA-1 at the CTL-target site (Kuhn, J. R., and Poenie, M. (2002)Immunity 16, 111-121), an indication that LFA-1 may possess a functionalrole other than strictly adhesion.

In conclusion we find that ICAM-2, as an LFA-1 ligand, can mediateactivation and clustering of the LFA-1 receptor—an event that in turnpolarizes the microtubule and actin cytoskeleton and activates thep44/42 MAPK pathway. These events were found to be necessary foreffector-target cell binding of CD56⁺CD8⁺ T cells, and perforin/granzymeA mediated cytolytic activity. This effect was shared by ICAM-3. Themechanisms governing LFA-1 receptor dynamics and intracellular signalingreported here suggest LFA-1 signaling functionally contributes inCD56⁺CD8⁺ cytolytic activity in addition to possessing an adhesive roleupon which other molecular interactions occur. Improper localization ofthe MTOC has been shown to inhibit exocytosis of lytic granules in CD8⁺tumor infiltrating T cells, thereby ablating perforin mediated cytolyticactivity necessary for a CTL response in murine tumor models (Radoja,S., at al., (2001) J Immunol 167, 5042-5051). Ironically, defective CD8⁺tumor infiltrating T cells can effectively mediate cell killing in vitro(Radoja, S., et al., (2001) J Immunol 166, 6074-6083), suggesting tumormediated inhibitory mechanisms exist within the tumor microenvironment.The production of soluble ICAMs (1 and 3) has been observed in sera fromcancer and autoimmunity patients, though analysis has not been extendedto ICAM-2 (Bloom, et al., (2002) J Rheumatol 29, 832-836). Only onereport has indicated that elevated levels of soluble ICAM-2 were presentin leukemia patients and decreased upon chemotherapy (Mustjoki, S., atal., (2001) Br J Haematol 113, 989-1000). The etiology of theseobservations is unknown. In the context of the work presented here, itis plausible to speculate that either dysregulation of surface ICAM-2 orsecretion of soluble ICAM-2 can prematurely trigger or block CD56⁺CD8⁺cytolytic activity at the effector-target site and permit tumor escapefrom T cell lysis. Other, specific roles, of ICAM-2 in its interactionwith other integrin ligands could lead to a better understanding ofevents that promulgate from the effector:target cell interface

Materials and Methods Immunological and Chemical Reagents

mAbs to b1, b2, b3, b4, b5, b6, a1, a4, a5, a_(L), LFA-1, Pyk2, SyK,Mac-1, ICAM-1, and ICAM-3 (PharMingen). CD3, CD4, CD8, CD19, CD56, CD45direct conjugates (FITC/PE/PERCP/APC/Biotin), granzyme-A-FITC(PharMingen). Perforin-CY5 and CD8-CY5PE (gift from the HerzenbergLaboratory, Stanford University). ICAM-2 mAb and ICAM-2-FITC (IC2/2Research Diagnostics). Anti-phospho PYK2(Y402), anti-phospho-p44/42(pT185Py187) (Biosource). Anti-phospho PKCa/b(Thr638),anti-phospho-Syk(Tyr525/526), anti-phosphoRaf1 (Ser259) (Cell SignalingTechnologies). Protein and chemical reagents used: fluoresceinisothiocyanate (FITC) (Pierce), Alexa fluor dye series 488, 546, 568,633, taxol-alexa546, phalloidin-alexa633, and CFSE (Molecular Probes).Tyrphostin A9 and 18, SB203580, piceatannol, bisindolylmaleimide I andII, herbimycin A (Calbiochem). Emodin, genistein, DMSO, PMA, PHA,staurosporine, ionomycin, propidium iodide, cytochalisin D (Sigma).Protein NG agarose (SCBT). Recombinant human IL-2 (Roche), recombinanthuman ICAM-1-FC, ICAM2-FC, ICAM3-FC (Genzyme). Secondary antibodies tomouse and rabbit IgG (Santa Cruz). Mock treatments consisted of mouseIgG (for antibodies), 1% BSA (for proteins), or 0.001% DMSO vehicle (forchemicals).

Cell Culture

NIH3T3 cells were maintained in DMEM, 10% DCS, 1% PSQ (DuelbeccoModified Eagle Media, 10% Donor calf serum, 1% penicillin-streptomycin(1000 units/ml and 2mM L-glutamine PSQ). Jurkat T-cells were maintainedin RPMI-1640, 10% FCS, 1% PSQ at 1×10⁵ cells/ml and serum starved 12hours for all functional assays. Cells were maintained at 5% CO₂/37° C.humidified incubator. Human peripheral blood monocytes were obtained byFicoll-plaque density centrifugation (Amersham Pharmacia, Uppsala,Sweden) of whole blood from healthy donors (Stanford Blood Bank) anddepleted for adherent cells. Magnetically activated cell sorting wasused to negatively isolate naïve CD8⁺ T cells for studies as indicated(Dynal, Oslo, Norway).

Soluble ICAM-2 Generation and Synthesis of ICAM-2-FITC and ICAM2-Beads

Full length ICAM2 cDNA was obtained from Jurkat cells and cloned intoretroviral vector PBM-Z-IN at the BamHI/Sal1 site as described (Perez ofal., 2002). Human ICAM-2 was overexpressed in NIH3T3 cells by retroviralinfection and harvested by immunoaffinity chromatography. ICAM-2 wasaffinity purified using a two step lysing procedure and subsequentpurification on an anti-ICAM-2 solid support. Cells were lysed in bufferA (20 mM Tris pH 7.5, 150 mM NaCl 1 mM EDTA 1 mM EGTA, 0.1% NP40, 2.5 mMNa₂PO₄, 1 mM b-glycerolphosphate, 1 mM Na₃VO₄ 1 mg/ml Leupeptin, 1 mMPMSF, protease inhibitor cocktail tablet (Boehringer Mannheim) for 5 min4° C., and subsequently permeabilized with 50% v/v with buffer B (BufferA plus 1% Triton-X-100) for 30 min 4° C. Supernatant was harvested bycentrifugation (14,000 RPM, 5 min, 4° C.). An Anti-ICAM-2 pAb to theC-terminal (4 mgs, Santa Cruz) was conjugated to an Affi-Gel Hzactivated support (Biorad) as suggested by manufacturer. This supportcouples Ig molecules via the FC region, resulting in higher antigenbinding capacity. Batch lysate of harvested supernatant was performed(4° C., for 2 hrs), and washed 4 times in buffer C (0.1% Tween-20, PBSpH 7.4). ICAM-2 protein was eluted by 4.5 M NaCl (in Tris pH 6.8),dialyzed overnight (in PBS pH 7.4, 0.001% azide, 0.01% glycerol, 4° C.),concentrated using size exclusion spin chromatography and stabilizedusing 0.01% glycerol. Anti-ICAM-2 solid support was re-equilibrated inbuffer C, stored in 0.001% thimerosol and re-used up to 3 times. Puritywas >98% as assessed by coomasie gel. Size exclusion chromatographyremoved higher molecular weight aggregates and were not observed onpurified ICAM-2 by native gel electrophoresis. 20 mgs were purified bythis method and used for this study. ICAM-2-FITC synthesis was achievedby chemical conjugation to NHS-Fluorescein (Pierce) and unreactive dyewas removed by gel filtration. ICAM-2-FITC probe did not integrate intotrypsinized Jurkat cells or bind when blocked by LFA-1 antibody clonesTS1/22 or TS1/18 (Developmental Hybridoma Studies Bank) or unlabeledICAM-2 protein as determined by flow cytometry. ICAM-2-FITC binding wasnot blocked by b2 integrin clone CT104 (Santa Cruz). Purified ICAM-2 wascomparable to human recombinant ICAM-2FC fusion proteih purified fromNSO murine myeloma cells (Genzyme). ICAM-1 FC and ICAM-3FC were alsopurified from NSO cells (Genzyme). Proteins were spun at 14,000 RPM, 5min prior to use. 1 mg of ICAM-2 protein was conjugated to 2×10⁸ epoxyactivated beads as suggested by manufacturer (Dynal). 4×10⁵ beadscontaining a total of 2 mg ICAM-2 protein were used as indicated. Gelimaging was performed on a VersaDoc machine (Biorad) and analyzed usingQuantity One quantitation software (Biorad).

Flow Cytometry

Intracellular and extracellular staining was performed as described(Perez and Nolan, 2002). Intracellular probes for active kinases weremade by conjugating phospho-specific antibodies to the Alexa Fluor dyeseries as described and used in phospho-protein optimized conditions(Perez and Nolan, 2002). Kinetic analyses was performed by directapplication of fixation buffer in time synchronized 96-wells maintainedat 37° C. Intracellular actin and microtubule staining was performedusing phalloidin-Alexa633 and taxol-Alexa546 dyes (Molecular Probes).Adhesion and clustering assays were performed using ICAM-2-FITC asdescribed in text. LFA-1 activation was assessed by eithermAb24-Alexa633 or mAb24-Alexa546 conjugate, surface stained at 37° C.Flow cytometry data are representative of 3 independent experiments of10⁸ cells/sample. 10-50,000 events were collected and manuallycalibrated on a FACSCalibur machine. Data plotted in bar graph format isexpressed as geometric mean fluorescence intensity (MFI) and normalizedfor isotype controls. Log ratios are defined as the MFI of stimulus tothe MFI of unstimulated cells. Data was analyzed using Flowjo software(Treestar).

Single Cell ICAM-2 Binding Measurements

Percentage of ICAM-2-FITC binding was expressed as100*((MFI_(exp)−MFI_(ctl))/(MFI_(final)−MFI_(ctl))), where MFI_(exp)equals the mean fluorescent intensity of experimental concentration,MFI, equals mean fluorescent intensity of unstained cells, MFI_(final)equals mean fluorescent intensity of final concentration that saturatedbinding. The samples were incubated with final concentrations asindicted in Figure for 30 min at 37° C. in 50 mL staining media (defRPMI, 4% FCS), washed 1X (500 mL, PBS pH 7.4, containing 1 mM EDTA), andresuspended in 100 mL (1% paraformaldehyde). Dilution factor of stainingconditions and molecular weight of 72.1 kD was used in determining molarconcentrations. The staining buffer contained 2.4 mM calcium and 2 mMmagnesium. The data were fit to the equation V=V_(max)[S]/(K_(m)+[S])where V is the percent bound, [S] is the ICAM-2-FITC concentration, andK_(m) is the Michaelis-Menten binding constant using Kaleidagraphsoftware.

Laser Scanning Confocal microscopy

Jurkat cells were treated as indicated and adhered to poly-L-lysine(Sigma) coated sterilized coverslips (1 mg/ml, 30 min) by mildcentrifugation (1000 RPM, 10 min), washed twice in phosphate bufferedsaline pH 7.4 (PBS) and fixed in 2.7% paraformaldehyde (in PBS). Cellswere permeabilized (5 min, 0.1% Triton-X-100 in PBS), washed twice inPBS, blocked in 4% FCS, and subjected to antibody or intracellularstaining as indicated. Stained coverslips were mounted and visualizedusing a Zeiss laser scanning confocal microscope 510.

Immunoprecipitations, Immunoblotting and Kinase Assays

Cell extracts were prepared by washing 2×10⁶ cells (treated asindicated) in ice cold PBS and harvesting in lysis buffer (20 mM Tris pH7.5, 150 mM NaCl 1 mM EDTA 1 mM EGTA, 1% Triton X-100, 2.5 mM Na₂PO₄, 1mM b-glycerolphosphate, 1 mM Na3VO₄, 1 mg/ml Leupeptin, 1 mM PMSF,protease inhibitor cocktail tablet (Boehringer Mannheim). Extracts werecentrifuged 14,000 RPM (5 min, 4° C.) and 10-20 mg (BCA protein assay(Pierce)) were immunoblotted using standard procedures.Immunoprecipitations (IP) were pre-cleared with protein A/G plus-agarosebeads, incubated with primary ab (1 h), protein A/G plus-agarose beads(1 h) and washed 4× with lysis buffer. Blots were incubated with theindicated antibodies and developed using ECL (Amersham). Immunoblotsstripped and reprobed (as indicated) were done by incubating withstripping buffer (62.5 mM Tris, pH 6.8, 10% SDS, 1% b-mercaptoethanol)(30 min, 55° C.). MAPK activity was detected by a p44/42 MAPK kinase kitas suggested by manufacturer (Cell Signaling Technologies).

Cytolytic Activity, Perforin Release Assays, and Conjugate FormationAssays

Target cell lysis was measured by flow cytoemtric based detection ofCFSE labeled HL60 cells. HL60 cells were labeled with 1 mg of CFSE (30min, 37° C.). Targets were washed twice and mock treated, IL-2 activated(100 U/ml), CD3/CD28 activated (1 mg/ml), or treated with ICAM2 beads orsoluble ICAM-1, -2, or 3 (10 mg/ml, 30 min, 37° C.) before plating at10⁴ target cells/well of a 96-well round bottom plate. CTLs were addedat 50:1, 25:1, and 12.5:1 E:T ratio, and incubated at 37° C. for 4 hrs.Cells were then processed for multiparameter flow cytometry andintracellular perforin stain. Percent specific lysis was calculated bythe following equation: % specific lysis=100-100×(experimental HL60count/total control HL60 count). HL60 counts were detectable by the CFSEfluorescence. Percent perforin was calculated by the following equation:% perforin=100×[(experimental perforin MFI−isotype mAb MFI)/(totalperforin MFI−isotype mAb MFI)]. MFI refers to mean fluorescent intensityof flow cytoemtric based intracellular detection. Cell conjugates weredetermined by flow cytometry as described (Morgan et al., 2001).Chemical inhibition was done at 10 mM of indicated compound (30 min, 37°C.) prior to stimulation as indicated. All experiments were performed intriplicate.

1. A method of detecting the clustering of receptor elements in singlecells, said method comprising: a) providing a sample comprising a cell,wherein said cell comprises receptor elements; b) contacting said cellwith a binding element that binds to at least one of said receptorelements to activate the formation of clusters, wherein said bindingelement comprises a label, wherein said clusters comprise i) at leasttwo of said receptor elements; ii) said binding element bound to atleast one of said receptor elements of said cluster; and, c) detectingsaid label.
 2. A method of simultaneously detecting multiple clusters insingle cells, said method comprising: a) providing a sample comprising acell wherein said cell comprises a plurality of different receptorelements; b) contacting said cell with a plurality of different bindingelements that bind to at least one of said different receptor elementsto activate the formation of multiple clusters, wherein each of saiddifferent binding elements comprise an identifying label, wherein saidclusters comprise i) at least two of said receptor elements; ii) atleast one of said binding elements bound to at least one of saidreceptor elements of said cluster; and c) detecting said label.
 3. Amethod of simultaneously detecting the activation state and clusteringof receptor elements in single cells, said method comprising: a)providing a sample comprising a cell wherein said cell comprises atleast first receptor elements; b) contacting said cell with at leastfirst activation-specific antibodies that binds to an isoform of saidfirst receptor elements, wherein each of said first activation-specificantibodies comprise an identifying label; c) contacting said cell withat least first binding elements that bind to at least one of saidreceptor elements to activate the formation of clusters, wherein each ofsaid first binding elements comprise an identifying label, wherein saidclusters comprise i) at least two receptor elements; ii) at least one ofsaid first binding elements bound to at least one of said receptorelements of said cluster; and d) simultaneously detecting saididentifying labels of said first activation-specific antibodies and saididentifying label of each of said clusters.
 4. The method according toclaim 1, wherein the binding of said binding elements to said receptorelements is activated by providing said cell with an activator.
 5. Themethod according to claim 1, wherein said label of said binding elementcomprises an antibody conjugated to a fluorophore, and wherein saidantibody binds to said binding element.
 6. The method according to claim1, wherein said clustering activates said receptor elements in saidclusters.
 7. The method according to claim 1, wherein said signal isproportional to the number of said clusters.
 8. The method according toclaim 1, wherein said detecting is by flow cytometry. 9-10. (canceled)11. The method according to claim 1, wherein said sample is from apatient. 12-13. (canceled)
 14. The method according to claim 2, whereinthe binding of said binding elements to said receptor elements isactivated by providing said cell with an activator.
 15. The methodaccording to claim 2, wherein said label of said binding elementcomprises an antibody conjugated to a fluorophore, and wherein saidantibody binds to said binding element.
 16. The method according toclaim 2, wherein said clustering activates said receptor elements insaid clusters.
 17. The method according to claim 2, wherein said signalis proportional to the number of said clusters.
 18. The method accordingto claim 2, wherein said detecting is by flow cytometry.
 19. The methodaccording to claim 2, wherein said sample is from a patient.
 20. Themethod according to claim 3, wherein the binding of said bindingelements to said receptor elements is activated by providing said cellwith an activator.
 21. The method according to claim 3, wherein saidlabel of said binding element comprises an antibody conjugated to afluorophore, and wherein said antibody binds to said binding element.22. The method according to claim 3, wherein said clustering activatessaid receptor elements in said clusters.
 23. The method according toclaim 3, wherein said signal is proportional to the number of saidclusters.
 24. The method according to claim 3, wherein said detecting isby flow cytometry.
 25. The method according to claim 3, wherein saidsample is from a patient.